Pentapeptide compounds and uses related thereto

ABSTRACT

Pentapeptide compounds are disclosed. The compounds have biological activity, e.g., cytotoxicity. Prodrugs having targeting groups and pentapeptide moieities, as well as precursors thereof are also disclosed. For example, precursors having a reactive linker that can serve as a reaction site for joining to a targeting agent, e.g., an antibody, as disclosed.

This application is the national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US02/13435 filed Apr. 30, 2002 which is a Continuation In Part of U.S. application Ser. No. 10/001,191, filed Nov. 1, 2001, and issued as U.S. Pat. No. 6,884,869, which is a Continuation In Part of U.S. application Ser. No. 09/845,786, filed Apr. 30, 2001, now abandoned, the disclosures of which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention is directed to biologically active organic compounds and precursors thereto, more specifically to pentapeptides and compositions containing these pentapeptides, and to methods for their use, e.g., as cytotoxic agents.

BACKGROUND OF THE INVENTION

Several short peptidic compounds have been isolated from natural sources and found to have biological activity. Analogs of these compounds have also been prepared, and some were found to have biological activity. For example, Auristatin E (Pettit, G. R., Barkoczy, J. “Tumor inhibiting tetrapeptide bearing modified phenethyl amides” U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine natural product, dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (Pettit, G. R. “The dolastatins” Prog. Chem. Org. Nat. Prod. 1997, 70, 1-79). Dolastatin 10, auristatin PE, and auristatin E are linear peptides comprised of four amino acids, three of which are unique to this class of compounds. Both dolastatin 10 and auristatin PE are in human clinical trials. The structural differences between the drugs reside in the C-terminal residue, in which the thiazolephenethyl amine of dolastatin 10 is replaced by a norephedrine unit in auristatin E.

The following references disclose dolastatin and auristatin compounds and analogs thereof

“Preparation of peptide derivatives as dolastatin 10 analogs with antitumor effects” Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi; Ito, Takeshi; Sugimura, Akihiro; Kobayashi, Motohiro. (Teikoku Hormone Mfg. Co., Ltd., Japan; Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi; Ito, Takeshi; Sugimura, Akihiro; Kobayashi, Motohiro). PCT International Patent Publication No. WO 9633212 A1 (1996);

“Preparation of dolastatin 10 analogs as cancer inhibitory peptides” Pettit, George R.; Srirangam, Jayaram K. (Arizona Board of Regents, USA). PCT International Patent Publication No. WO 9614856 A1 (1996);

“Preparation of tetra- and pentapeptide dolastatin analogs as anticancer agents” Pettit, George R.; Srirangam, Jayaram K.; Williams, Michael D. (Arizona Board of Regents, USA). Eur. Pat. Appl. No. EP 695757 A2 (1996);

“Preparation of dolastatin 10 analog pentapeptide amides and esters as anticancer agents” Pettit, George R.; Srirangam, Jayaram K. (Arizona Board of Regents, USA). Eur. Pat. Appl. No. EP 695758 A2 (1996);

“Preparation of dolastatin analog pentapeptide methyl esters as anticancer agents” Pettit, George R.; Williams, Michael D.; Srirangam, Jayaram K. (Arizona Board of Regents, USA). Eur. Pat. Appl. No. EP 695759 A2 (1996);

“Preparation of novel peptide derivative as antitumor agent” Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi; Ito, Takeshi; Sugimura, Akihiro; Kobayashi, Motohiro. (Teikoku Hormone Mfg. Co., Ltd., Japan). PCT International Patent Publication No. WO 9509864 A1 (1995);

“Preparation of tetrapeptide derivatives as antitumor agents” Sakakibara, Kyoichi; Gondo, Masaaki; Miyazaki, Koichi. (Teikoku Hormone Mfg. Co., Ltd., Japan). PCT International Patent Publication No. WO 9303054 A1 (1993);

“Antineoplastic agents 365. Dolastatin 10 SAR probes” Pettit, George R.; Srirangam, Jayaram K.; Barkoczy, Jozsef; Williams, Michael D.; Boyd, Michael R.; Hamel, Ernest; Pettit, Robin K.; Hogan, Fiona; Bai, Ruoli; Chapuis, Jean-Charles; McAllister, Shane C.; Schmidt, Jean M., Anti-Cancer Drug Des. (1998), 13(4), 243-277;

“Antineoplastic agents. 337. Synthesis of dolastatin 10 structural modifications” Pettit, George R.; Srirangam, Jayaram K.; Barkoczy, Jozsef; Williams, Michael D.; Durkin, Kieran P. M.; Boyd, Michael R.; Bai, Ruoli; Hamel, Ernest; Schmidt, Jean M.; Chapius, Jean-Charles, Anti-Cancer Drug Des. (1995), 10(7), 529-44; and

“Synthesis and antitumor activity of novel dolastatin 10 analogs” Miyazaki, Koichi; Kobayashi, Motohiro; Natsume, Tsugitaka; Gondo, Masaaki; Mikami, Takashi; Sakakibara, Kyoichi; Tsukagoshi, Shigeru, Chem. Pharm. Bull. (1995), 43(10), 1706-18.

There is a need in the art for improved antitumor agents. Despite promising in vitro data for dolastatin 10 and its analogs, significant general toxicities at doses required for achieving a therapeutic affect compromise their efficacy in clinical studies. The present invention is directed to fulfilling this need and provides further advantages as disclosed herein.

The recitation of any reference in Section 2 of this application is not an admission that the reference is prior art to this application.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl; and

R⁹ is selected from

R¹⁰ is selected from

R¹¹ is selected from hydrogen and lower alkyl;

R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence;

R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene;

R¹⁵ is selected from hydrogen, lower alkyl and aryl;

R¹⁶ is selected from arylene(lower alkylene), lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and

R¹⁷ is selected from

where Y=O or S.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl;

R¹¹ is selected from hydrogen and lower alkyl;

R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and

R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety, where R²⁰ can be bonded to the carbon labeled “x” by either a single or double bond.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl;

R¹¹ is selected from hydrogen and lower alkyl;

R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and

R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety, where R²⁰ can be bonded to the carbon labeled “x” by either a single or double bond.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n) wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl; and

R²⁰ is a reactive linker group comprising a reactive site that allows R²⁰ to be reacted with a targeting moiety.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl;

R¹¹ is selected from hydrogen and lower alkyl; and

R¹⁸ is selected from hydrogen, a hydroxylprotecting group, and a direct bond where OR¹⁸ represents ═O.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl; and

R¹⁹ is selected from hydroxy- and oxo-substituted lower alkyl.

In another aspect, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof

wherein, independently at each location:

R¹ is selected from hydrogen and lower alkyl;

R² is selected from hydrogen and lower alkyl;

R³ is lower alkyl;

R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6;

R⁶ is selected from hydrogen and lower alkyl;

R⁷ is sec-butyl or iso-butyl;

R⁸ is selected from hydrogen and lower alkyl; and

R⁹ is selected from

R¹⁰ is selected from

R¹¹ is selected from hydrogen and lower alkyl;

R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence;

R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; and

R¹⁵ is selected from hydrogen, lower alkyl and aryl.

Optionally, R⁹ is

and R¹⁰ is

Optionally, R⁹ is

and R¹⁰ is

Optionally, R⁹ is

and R¹⁰ is

Optionally, R⁹ is

and R¹⁰ is

In another aspect, the present invention provides a composition comprising a biologically active compound as described above or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the present invention provides a method for killing a cell, the method comprising administering to the cell a lethal amount of a compound, a pharmaceutically acceptable salt and solvate thereof, or composition as described above.

In another aspect, the present invention provides a method of killing a cell comprising

a. delivering a compound, a pharmaceutically acceptable salt or solvate thereof, or composition as described above to a cell, where the compound or pharmaceutically acceptable salt or solvate thereof enters the cell;

b. cleaving mAb from the remainder of the compound or pharmaceutically acceptable salt or solvate thereof; and

c. killing the cell with the remainder of the compound or pharmaceutically acceptable salt or solvate thereof.

In another aspect, the present invention provides a method of killing or inhibiting the multiplication of tumor cells or cancer cells in a human or other animal, the method comprising administering to the human or animal a therapeutically effective amount of a compound, pharmaceutically acceptable salt or solvate thereof, or composition described above.

In still another aspect, the present invention provides methods for killing or inhibiting the multiplication of a cell comprising:

a. delivering a compound of the invention, a pharmaceutically acceptable salt or solvate thereof, or composition as described above to a cell, where the compound enters the cell;

b. cleaving the antibody from the remainder of the compound or pharmaceutically acceptable salt or solvate thereof; and

c. killing the cell with the remainder of the compound or pharmaceutically acceptable salt or solvate thereof.

In yet another aspect, the present invention provides a method of killing or inhibiting the multiplication of tumor cells or cancer cells in a human or other animal, the method comprising administering to a human or other animal in need thereof an effective amount of a compound, pharmaceutically acceptable salt of the compound or composition described above.

In another aspect, the invention provides methods for treating cancer, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition, described above.

In still another aspect, the invention provides a method of killing or inhibiting the multiplication of cells that produce auto-immune antibodies, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition, described above.

In yet another aspect, the invention provides methods for treating an autoimmune disease, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition, described above.

In still another aspect, the invention provides methods for treating an infectious disease, comprising administering to a human or other animal in need thereof an effective amount of a compound, a pharmaceutically acceptable salt or solvate thereof or composition described above.

These and related aspects of the present invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a synthetic scheme illustrating the preparation of specific tripeptides useful in preparing compounds of the present invention.

FIG. 2 shows a synthetic scheme illustrating the preparation of specific dipeptides useful in preparing compound of the present invention.

FIGS. 3, 4 and 5 show synthetic schemes illustrating the preparation of pentapeptides from tripeptides and dipeptides

FIGS. 6, 7 and 8 show synthetic schemes for conjugating a pentapeptide to a heterobifunctional linker to provide ligand (specifically a mAb) reactive drug molecules.

FIGS. 9, 10, 11 and 12 show synthetic schemes for conjugating a ligand, and specifically a mAb, to a drug-reactive linker conjugate to provide prodrugs of the present invention.

FIG. 13 shows the synthetic scheme used for the preparation of compound 49.

FIG. 14 shows a synthetic scheme for conjugating a pentapeptide to a heterobifunctional linker to provide drug-reactive linker conjugate 51.

FIG. 15 shows the synthetic scheme used for the preparation of compound 53.

FIG. 16 shows a synthetic scheme illustrating the preparation of a specific dipeptide (57) useful in preparing compounds of the present invention.

FIG. 17 shows a synthetic scheme for conjugating a pentapeptide (58) to a heterobifunctional linker to provide drug-reactive linker conjugate 59.

FIGS. 18A, 18B and 18C show the in vitro cytotoxicity of drugs and mAb-drug conjugates on (A) and (B) L2987 human lung adenoma cells, and (A) and (C) Kato III human gastric carcinoma cells.

FIGS. 19A and 19B show the cytotoxic effects of auristatin E and auristatin E-containing conjugates on L2987 human lung adenocarcinoma cells (FIG. 19A) and various hematologic cell lines treated with AC10-S-40a (FIG. 19B).

FIGS. 20A and 20B show median tumor volume observed when nude mice with subcutaneous L2987 human lung adenocarcinoma or 3396 human breast carcinoma xenografts were injected with conjugates or drug according to the schedule shown in the two Figures.

FIG. 21 shows the cytotoxic effects of drug conjugates of the invention on H3396 human breast carcinoma cells.

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the present invention provides short peptide-containing compounds having biological activity, e.g., cytotoxicity, and methods for their use, e.g., in cancer therapy. Before describing the compounds of the present invention, reference is made to the following definitions that are used herein.

Definitions

As used herein the following terms have the indicated meanings:

“Animal subjects” include humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, and cats. Birds, including fowl, are also a suitable animal subject.

“Aryl” refers to aromatic groups which have at least one ring having a conjugated pi electron system and includes carbocyclic aryl and heterocyclic aryl. In specific aspects of the invention, the aryl group (and the arylene group as discussed below) has 5-14, or 6-12, or 6-10 atoms as part of one or more aromatic rings. In one aspect the aryl or arylene is a carbocyclic. “Carbocyclic aryl” refers to aromatic groups wherein the ring atoms of the aromatic ring are all carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic groups, all of which may be optionally substituted. Exemplary carbocyclic aryl groups include phenyl, naphthyl and anthracenyl. Exemplary substituted carbocyclic aryl groups include indene and phenyl substituted by one or two substituents such as, or selected from, lower alkyl, hydroxy, lower alkoxy, lower alkoxycarbonyl, halogen, trifluoromethyl, nitro, and cyano. “Heterocyclic aryl”, which may also be called “heteroaryl” refers to aryl groups having from 1 to 9 carbon atoms and the remainder of the atoms are heteroatoms, and includes those heterocyclic systems described in “Handbook of Chemistry and Physics,” 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber Co., Cleveland, Ohio. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental Heterocyclic Systems. Suitable heteroatoms include oxygen, nitrogen, and S(O)_(i), wherein i is 0, 1 or 2. Suitable examples of heteroaryl groups include, without limitation, benzofuran, benzothiophene, indole, benzopyrazole, coumarin, isoquinoline, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, triazole, quinoline, pyrimidine, pyridine, pyridone, pyrazine, pyridazine, isothiazole, isoxazole and tetrazole. “Aryl groups” are bonded to one other group, while “arylene” refers to an aryl group that is bonded to two other groups. For example, phenyl

is an aryl group, while para-phenylene

is one example of an arylene group. Other arylene groups are ortho- or meta-phenylene and naphthalene.

“Arylene (lower alkylene)” refers to a group formed from an arylene group and an alkylene group, that together link two other groups. For example, in X-arylene(lower alkylene)-Y the arylenealkylene group links X and Y, and is formed from a lower alkylene, e.g., methylene (—CH₂—) and an arylene (e.g.,

to provide the aryl (lower alkylene), e.g.,

“Carbocycle” or “carbocyclic ring” refers to any saturated, unsaturated or aromatic monocyclic ring where the ring atoms are carbon. A C₅₋₇carbocycle refers to any carbocyclic ring having 5, 6 or 7 carbon atoms that form the ring. Examples of such carbocycles include, but are not limited to, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, and phenyl.

“Compound”, as in the terms “compound of the formula”, “compound of the structure”, “compound of the invention”, and the like, shall refer to and encompass the chemical compound itself as well as, whether explicitly stated or not, and unless the context makes clear that the following are to be excluded: amorphous and crystalline forms of the compound, including polymorphic forms, where these forms may be part of a mixture or in isolation; free acid and free base forms of the compound, which are typically the forms shown in the structures provided herein; isomers of the compound, which refers to optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and the optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; where an isomer may be in isolated form or in admixture with one or more other isomers; isotopes of the compound, including deuterium- and tritium-containing compounds, and including compounds containing radioisotopes, including therapeutically- and diagnostically-effective radioisotopes; multimeric forms of the compound, including dimeric, trimeric, etc. forms; salts of the compound, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts having organic counterions and inorganic counterions, and including zwitterionic forms, where if a compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates of the compound, including hemisolvates, monosolvates, disolvates, etc., including organic solvates and inorganic solvates, said inorganic solvates including hydrates; where if a compound is associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some instances, reference made herein to a compound of the invention will include an explicit reference to one or of the above forms, e.g., salts and solvates, however, this reference is for emphasis only, and is not to be construed as excluding other of the above forms as identified above.

“Direct bond” refers to a pair of electrons that hold together two adjacent atoms. Thus, when two atoms are separated by a direct bond, those two atoms are bonded together by a covalent bond. If a direct bond is attached to only one atom (X) and X is bonded to an adjacent atom (Y), then the direct bond represents a second bond between X and Y, i.e., the direct bond option provides a double bond between X and Y, at the expense of a hydrogen that would otherwise be bonded to Y. For example C—O—R where R is a direct bond denotes C═O.

“Hydroxylprotecting group” includes, but is not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethyl silyl ether, acetate ester, substituted acetate esters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Independently at each location” and “independently at each occurrence” are synonymous terms which mean that any selection is independent of any other selection. For instance, if a variable is selected from two options, and the variable occurs twice, then the option selected at one occurrence of the variable does not impact in any way the choice of the option selected at the second occurrence of the variable.

“Leaving group” refers to a functional group that can be readily substituted by another functional group. A preferred leaving group is replaced by S_(N)2 displacement by a nucleophile. Such leaving groups are well known in the art, and include halides (e.g., chloride, bromide, iodide), methanesulfonate (mesylate), p-toluenesulfonate (tosylate), trifluoromethane sulfonate and trifluoromethylsulfonate.

“Lower alkyl” refers to a straight or a branched chain, cyclic or acyclic, monovalent saturated hydrocarbon, halocarbon or hydrohalocarbon radical of one to ten carbon atoms. In various aspects, the lower alkyl group has 1-8,1-6, 1-5, or 1-4 carbons. Thus, the alkyl group may optionally have halogen substitution, where a halocarbon contains only carbon and halogen, and where a hydrohalocarbon contains carbon, halogen and hydrogen. Exemplary lower alkyl groups are, without limitation, methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. “Lower alkylene” refers to a lower alkyl group that is bonded to two groups, such as (—CH₂—)₁₋₈, (—CH₂—)₁₋₆, (—CH₂—)₁₋₅ or (—CH₂—)₁₋₄. For example, methyl (—CH₃) is a lower alkyl group, while methylene (—CH₂—) is a lower alkylene group. A hydroxy- or oxo-substituted lower alkyl refers to a lower alkyl group wherein a hydrogen on the lower alkyl group is replaced by —OH (for a hydroxy-substituted lower alkyl), or two hydrogens on a single carbon of the lower alkyl group are replaced by ═O (for an oxo-substituted lower alkyl).

The term “antibody,” as used herein, refers to an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce auto-immune antibodies associated with an autoimmune disease. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Antibodies used in the invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, sFvs, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens and microbial antigens.

The shorthand designation “mAb” is used in chemical structures herein to encompass any antibody, i.e., any immunoglobin molecule or any immunologically active portion of an immunoglobin molecule.

As used herein, the term “viral antigen” includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuramimidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) which is capable of eliciting an immune response. As used herein, the term “microbial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein saccharaide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide ⅝) which is capable of eliciting an immune response.

“Pharmaceutically acceptable salt” and “salts thereof” refers to organic or inorganic salts of the pharmaceutically important molecule. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions. Hence, the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion. The desired charge distribution is determined according to methods of drug administration. Examples of pharmaceutically acceptable salts are well known in the art but, without limiting the scope of the present invention, exemplary presentations can be found in the Physician's Desk Reference, The Merck Index, The Pharmacopoeia and Goodman & Gilman's The Pharmacological Basis of Therapeutics. The salt will typically be either an acid addition salt, i.e., a salt formed by adding an acid to a pentapeptide compound, or a base addition salt, i.e., a salt formed by adding a base to a pentapeptide compound. The pentapeptide compounds of the invention contain at least one amine group, and accordingly acid addition salts may be formed with this amine group, where acid addition salts refer to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, isonicotinic, lactic, salicylic, oleic, tannic, pantothenic, ascorbic, succinic, gentisinic, fumaric, gluconic, glucuronic, formic, glutamic, benzenesulfonic, pamoic acid, and the like.

Compounds included in the present compositions that include an amino moiety may form pharmaceutically or cosmetically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds, included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, and iron salts.

“Pharmaceutically acceptable solvate” refers to a solvate, i.e., an association of a solvent and a compound of the invention, that retains the biological effectiveness and properties of the biologically active pentapeptide compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. It will be appreciated by those skilled in the art that solvated forms are biologically equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Sykes, R. A., Guidebook to Mechanism in Organic Chemistry, 6^(th) Ed (1986, John Wiley & Sons, N.Y.) is an exemplary reference that describe solvates.

In the context of cancer, the term “treating” includes any or all of: inhibiting tumor growth, inhibiting replication of tumor cells, lessening of overall tumor burden and ameliorating of one or more symptoms associated with the disease.

In the context of an auto immune disease, the term “treating” includes any or all of: inhibiting replication of cells capable of producing an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includes any or all of: inhibiting replication of an infectious agent, lessening the infectious agent burden and ameliorating one or more symptoms of an infectious disease.

The following abbreviations may be used herein and have the indicated definitions: t-Boc is tert-butoxy carbonyl, DCC is dicyclohexylcarbodiimide, DIAD is diisopropylazodicarboxylate; Fmoc is 9-fluorenylmethoxycarbonyl, DEPC is diethyl phosphorocyanidate, DMAP is 4-(N,N-dimethylamino) pyridine, PyBrop is bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, TEAA is triethylammonium acetate, TFA is trifluoroacetic acid, and Z is benzyloxycarbonyl.

In the phrase “R²⁰ is a reactive linker having a reactive site that allows R²⁰ to be reacted with a targeting moiety”, a “reactive site” refers to a functional moiety that can undergo reaction with a ligand so as to provide a covalent bond between the ligand and the reactive linker. In one aspect of the invention, the reactive site is reactive with a functional group selected from thiol, amino and hydroxyl, preferably, thiol and amino. Both thiol and amino groups are present in proteins, where thiol groups are, e.g., produced by reduction of disulfide bonds in proteins, and amino groups are present in, e.g., a lysine moiety of a protein.

In a preferred aspect of the invention, the reactive linker has a reactive site that is reactive with a thiol group. For instance, the reactive site may be a maleimide of the formula

where the carbon-carbon double bond is reactive with nucleophiles, e.g., thiol groups. Alternatively, the reactive site may have the formulae

where X is a leaving group that may be displaced by a nucleophile, e.g., a thiol group as found on a protein.

In another preferred aspect of the invention, the reactive linker contains a reactive site that is reactive with an amine or amino group. Suitable amine reactive sites include, without limitation, activated esters such as N-hydroxysuccinimide esters, p-nitrophenyl esters, pentafluorophenyl esters, and other reactive functionalities, such as isothiocyanates, isocyanates, anhydrides, acid chlorides, and sulfonyl chlorides. Each of these functionalities provides a reactive site for a reactive linker of the present invention.

Many compounds of the invention have a norephedrine moiety of the following general formula:

In one embodiment, the compounds' norephedrine moiety has (1S, 2R) stereochemistry as depicted below:

In alternate embodiments, the compounds' norephedrine moiety has (1S, 2S), (1R, 2S) or (1R, 2R) stereochemistry.

Compounds

In one aspect, the present invention provides compounds of the general structure “drug-linker-targeting agent”, where the drug is a pentapeptide as disclosed herein and the targeting agent is an antibody. As is understood by those skilled in the art, such compounds are generally known as antibody conjugates or antibody therapeutic-agent conjugates. Such compounds have the following structure, and may also be referred to herein as prodrugs.

(and pharmaceutically acceptable salts and solvates thereof)

In this structure, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; and R⁹ is selected from

wherein R¹⁰ is selected from

R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and R¹³ is

wherein: R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; R¹⁵ is selected from hydrogen, lower alkyl and aryl; R¹⁶ is selected from arylene(lower alkylene), lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is selected from

where Y=O or S.

In optional embodiments of the invention: R¹ is hydrogen; R¹ and R² are methyl; R³ is isopropyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle and R⁵ is selected from H and methyl; R⁴ is selected from lower alkyl, and R⁵ is selected from H and methyl; R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is lower alkyl; R⁸ is hydrogen.

In another optional embodiment, R⁹ is

and R¹⁰ is selected from

In one embodiment R¹⁰ is

while in another embodiment R¹⁰ is

In a preferred embodiment, R¹⁴ is selected from arylene and lower alkylene; R¹⁵ is selected from lower alkyl and aryl; and R¹⁶ is lower alkylene.

In another optional embodiment, R⁹ is

and R¹⁰ is selected from

In a preferred embodiment, R¹⁴ is selected from arylene and lower alkylene; R¹⁵ is selected from lower alkyl and aryl; and R¹⁶ is lower alkylene.

In another optional embodiment, R⁹ is

and R¹³ is

In a preferred embodiment, R¹⁵ is lower alkyl; and R¹⁶ is lower alkylene.

In another optional embodiment, R⁹ is

In a preferred embodiment, R¹⁶ is lower alkylene.

In another optional embodiment, R⁹ is

In a preferred embodiment, R¹⁶ is selected from lower alkylene and arylene.

In one embodiment R¹⁷ is

In another embodiment, R¹⁷ is

In another embodiment, R¹⁷ is

where Y=O or S.

For example, the present invention provides a prodrug of the formula

and pharmaceutically acceptable salts and solvates thereof.

In addition, the present invention provides a prodrug of the formula:

In addition, the present invention provides a prodrug of the formula:

and pharmaceutically acceptable salts and solvates thereof.

In addition to prodrugs as described above, the present invention also provides intermediate compounds that can be used to prepare prodrugs. These intermediate compounds contain a reactive linker group R²⁰ having a reactive site that allows R²⁰ to be reacted with a targeting moiety, e.g., an mAb.

In one aspect, the invention provides intermediate compounds of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety, where R²⁰ can be bonded to the carbon labeled “x” by either a single or double bond.

In another aspect, the present invention provides intermediate compounds of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety, where R²⁰ can be bonded to the carbon labeled “x” by either a single or double bond.

In another aspect, the present invention provides intermediate compounds of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; and R²⁰ is a reactive linker group comprising a reactive site that allows R²⁰ to be reacted with a targeting moiety.

In any of the foregoing intermediate compounds, in further aspects R²⁰ comprises a hydrazone of the formula

wherein: R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; R¹⁵ is selected from hydrogen, lower alkyl and aryl; and R¹⁶ is selected from arylene(lower alkylene), lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5.

Additionally, in any of the foregoing intermediate compounds, in further aspects R²⁰ comprises a hydrazone of the formula

wherein: R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; R¹⁵ is selected from hydrogen, lower alkyl, and aryl; and R¹⁶ is selected from arylene(lower alkylene), lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5.

Additionally, in any of the foregoing intermediate compounds, in further aspects R²⁰ comprises a hydrazone of the formula:

wherein: R¹⁶ is selected from lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5, and x identifies the carbon also marked x in the intermediate structures shown above.

As mentioned previously, a reactive linker group R²⁰ includes a reactive site that can be reacted with a targeting moiety so as to indirectly join the targeting moiety to the drug. The following are exemplary reactive sites that may be present as part of the R²⁰ group. For example, any of the following reactive sites may be joined to any of the hydrazone structures set forth above in order to form a complete R²⁰ structure. In one aspect, R²⁰ comprises a reactive site having the formula

In another aspect, R²⁰ comprises a reactive site having the formula

wherein X is a leaving group. In another aspect, R²⁰ comprises a reactive site having the formula

wherein X is a leaving group.

For example, the present invention provides intermediate compounds of the formulae:

and pharmaceutically acceptable salts and solvates thereof wherein R⁴ is selected from iso-propyl and sec-butyl, and R⁵ is hydrogen;

and pharmaceutically acceptable salts and solvates thereof.

In addition to prodrugs as described above, and direct precursors thereto as described above and referred to as intermediate compounds, the present invention also provides precursors to the intermediate compounds. In one aspect of the invention, these precursors to the intermediate compounds have a carbonyl group. As discussed in more detail below, a carbonyl group is conveniently reacted with hydrazine derivatives in order to prepare intermediate compounds having R²⁰ groups and reactive sites as part of the R²⁰ groups. Thus, in a further aspect, the present invention provides the following precursors to the intermediate compounds, where these precursors have either carbonyl groups or hydroxyl groups that could be readily oxidized to a carbonyl group.

In one aspect, the present invention provides compounds of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; and R¹⁹ is selected from hydroxy- and oxo-substituted lower alkyl. In various optional embodiments: R¹ is hydrogen; or R¹ and R² are methyl; and/or R³ is iso-propyl; and/or R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle and R⁵ is selected from H and methyl; or R⁴ is selected from lower alkyl, and R⁵ is selected from H and methyl; or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; and/or R⁶ is lower alkyl; and/or R⁸ is hydrogen; and/or R¹⁹ is oxo-substituted lower alkyl. An exemplary compound of this aspect of the invention is

and pharmaceutically acceptable salts and solvates thereof.

In another aspect the present invention provides compounds of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R⁹ is selected from

R¹⁰ is selected from

R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; and R¹⁵ is selected from hydrogen, lower alkyl and aryl. In various embodiments of this aspect of the invention: R¹⁰ is

or R¹⁰ is

Some specific compounds of this aspect of the invention have the structures

and pharmaceutically acceptable salts and solvates thereof

wherein R⁴ is iso-propyl or sec-butyl and R⁵ is hydrogen.

In a further aspect the present invention provides pentapeptide drugs of the following structures. In these structures the drug may contain a carbonyl group that could optionally be reacted directly with a hydrazine derivative or other reactive group so as to introduce an R²⁰ group into the molecule. Thus, in one embodiment, the present invention provides a pentapeptide drug of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; and R¹⁸ is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR¹⁸ represents ═O. For instance, the present invention provides compound of the formula

and pharmaceutically acceptable salts and solvates thereof.

In another embodiment, the present invention provides a pentapeptide drug of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; and R¹⁸ is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR¹⁸ represents ═O. For instance, the present invention provides compounds of the formulae

and pharmaceutically acceptable salts and solvates thereof wherein R⁴ iso-propyl and R⁵ is hydrogen.

In another embodiment, the present invention provides a pentapeptide drug of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; and R¹⁹ is selected from hydroxy- and oxo-substituted lower alkyl. For instance, the present invention provides a compound of the formula

and pharmaceutically acceptable salts and solvates thereof.

In various further embodiments, the pentapeptide drugs of the invention have one of the three general pentapeptide structure as shown above, wherein: R¹ is hydrogen; or R¹ and R² are methyl; and/or R³ is isopropyl; and/or R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle and R⁵ is selected from H and methyl; or R⁴ is selected from lower alkyl, and R⁵ is selected from H and methyl; or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; and/or R⁶ is lower alkyl; and/or R⁸ is hydrogen; and/or R¹¹ is hydrogen; and/or —OR¹⁸ is ═O; and/or R¹⁹ is oxo-substituted lower alkyl.

General Synthesis

In various aspects as detailed above, the present invention provides drug-linker-ligand conjugates as well as precursors thereto. A drug-linker-ligand conjugate of the present invention is designed so that upon delivery of the conjugate to a cell, and particularly a tumor cell, the conjugate will partially degrade in a manner that frees the drug, from the ligand with perhaps some residue from the linker remaining attached to the drug, and typically with some residue of the linker remaining attached to the ligand. The drug (or drug-linker residue) will then act in a cytotoxic manner to kill the tumor cells, or at least to mitigate the multiplication of the tumor cells. The drug-linker-ligand conjugate may be referred to herein as a prodrug. The ligand is typically a targeting moiety that will localize the prodrug in the vicinity of a tumor cell.

The prodrug has the basic structure shown below:

and pharmaceutically acceptable salts and solvates thereof where the—linker-ligand portion of the prodrug is joined to the drug at any of positions a, b, or c. As described in more detail below, such conjugates are conveniently prepared using a heterobifunctional linker. In a preferred aspect of the invention, the heterobifunctional linker includes, on one end, a thiol acceptor i.e., a chemical moiety that will react with a thiol group so that the sulfur atom of the thiol group becomes covalently bonded to the thiol acceptor. Thiol acceptors are well known in the art, where exemplary thio acceptor group are maleimide and haloacetamide groups. The thiol acceptor group provides a convenient site for ligand attachment. At the other end, in a preferred aspect the heterobifunctional linker has a hydrazide group. Hydrazide groups are useful endgroups for a heterobifunctional linker because they readily react with carbonyl groups, i.e., aldehyde and ketone groups, where carbonyl groups can be readily incorporated into a pentapeptide compound as disclosed herein, to provide a hydrazone linkage. Incorporation of a hydrazone into the conjugate is particularly preferred in order to impart desired pH-sensitivity to the conjugate. Thus, under low pH conditions, as in acidic intracellular compartments (e.g., lysosomes), a hydrazone group will cleave and release the drug free from the ligand. A preferred linker is an acid-labile linker, that is cleaved under low pH conditions, i.e., conditions where the pH is less than about 6, preferably less than about 5.

The drugs of the present invention may be generally described as pentapeptides. Typically, pentapeptide drugs of the present invention can be prepared by introduction of a peptide bond between selected amino acids and/or modified amino acids, and/or peptide fragments. Such peptide bonds may be prepared, for example, according to the liquid phase synthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.

A preferred method to prepare pentapeptide drugs of the present invention entails preparing a dipeptide and a tripeptide, and combining stoichiometric equivalents of these two fragments in a one-pot reaction under suitable condensation conditions. This approach is illustrated in the following Schemes 1-3. Thus, the tripeptide 6 may be prepared as shown in Scheme 1, and the dipeptide 9 may be prepared as shown in Scheme 2. The two fragments 6 and 9 may be condensed to provide a pentapeptide (10) as shown in Scheme 3.

As illustrated in Scheme 1, a PG-protected amino acid 1 (where PG represents an amine protecting group, e.g., benzyloxycarbonyl (which is a preferred PG and is represented herein by Z), R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocyclic when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocyclic group of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6) is coupled to t-butyl ester 2 (where R⁶ is selected from hydrogen and lower alkyl; and R⁷ is sec-butyl or iso-butyl) under suitable coupling conditions, e.g., in the presence of PyBrop and diisopropylethylamine, or using DCC (as accomplished, for example, by Miyazaki, K. et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).

Suitable protecting groups PG, and suitable synthetic methods to protect an amino group with a protecting group are well known in the art. See, e.g., Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd Edition, 1991, John Wiley & Sons. Preferred protected amino acids 1 are PG-Ile and, particularly, PG-Val, while other suitable protected amino acids include, without limitation: PG-cyclohexylglycine, PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid, PG-α-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, and PG-tert-butylglycine. Z, i.e., benzyloxycarbonyl, is a preferred protecting group. Fmoc is another preferred protecting group. A preferred t-butyl ester 2 is dolaisoleulne t-butyl ester.

After chromatographic purification, the dipeptide 3 is deprotected, e.g., using H₂ and 10% Pd—C in ethanol when PG is benzyloxycarbonyl, or using diethylamine for removal of an Fmoc protecting group. The resulting amine 4 readily forms a peptide bond with an amino acid 5 (where R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; and R³ is lower alkyl). N,N-Dialkyl amino acids are preferred amino acids 5, such as commercially available N,N-dimethyl valine. Other N,N-dialkyl amino acids can be prepared by reductive bis-alkylation following known procedures (see, e.g., Bowman, R. E, Stroud, H. H, J. Chem. Soc., 1950, 1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two preferred amino acids 5 for the synthesis of N-monoalkyl derivatives. The amine 4 and the amino acid 5 are conveniently joined to provide the tripeptide 6 using coupling reagent DEPC with triethylamine as the base.

The dipeptide 9 can be readily prepared by condensation of the modified amino acid t-Boc-Dolaproine 7 (see, for example, Pettit, G. R., et al. Synthesis, 1996, 719-725), with commercially available (1S, 2R)-norephedrine, L- or D-phenylalaminol, or with synthetic p-acetylphenethylamine 8 (Shavel, J., Bobowski, G., 1969, U.S. Pat. No. 3,445,518) using condensing agents well known for peptide chemistry, such as, for example, DEPC in the presence of triethylamine, as shown in Scheme 2.

Scheme 3 illustrates the joining together of the tripeptide 6 with the dipeptide 9 to form a pentapeptide 10. The joining together of these two fragments can be accomplished using, e.g., TFA to facilitate Boc and t-butyl ester cleavage, respectively, followed by condensation conditions, e.g., utilizing DEPC, or similar coupling reagent, in the presence of excess base (triethylamine or equivalent) to give the desired product in moderate but acceptable yields.

To obtain N-monoalkyl pentapeptide 10, the Fmoc group should be removed from the N-terminal amino acid after final coupling by treatment with diethylamine (see General Procedure E described below).

Upon proper selection of a, b, and c, the present invention provides pentapeptides of the formula

and pharmaceutically acceptable salts and solvates thereof which are drugs of the present invention upon appropriate selection of a, b, and c, where these drugs may be prepared as described above. Thus, as referred to herein, the drugs of the present invention are not necessarily the same as the degradation product of the prodrugs of the present invention. Rather, the drugs of the present invention are pharmacologically active, cytotoxic chemicals of the above structure, which are conveniently utilized to prepare prodrugs of the present invention, where these prodrugs will release a pharmacalogically active agent upon partial degradation of the prodrug.

Thus, in one aspect, the present invention provides drugs having the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; and R¹⁸ is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR¹⁸ represents ═O.

In another aspect, the present invention provides drugs having the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; and R¹⁸ is selected from hydrogen, a hydroxyl protecting group, and a direct bond where OR¹⁸ represents ═O.

In another aspect, the present invention provides drugs having the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; and R¹⁹ is selected from hydroxy- and oxo-substituted lower alkyl.

In order to prepare a prodrug of the present invention, the drug is either directly reacted with a linker, typically a heterobifunctional linker, or else the drug is somewhat modified in order that it contains a reactive site that is suitably reactive with a reactive site on a heterobifunctional linker. In general, the heterobifunctional linker has the structure

In a preferred embodiment of the invention, Reactive Site No. 1 is reactive with a carbonyl group of the drug, Reactive Site No. 2 is reactive with functionality on the ligand, and Reactive Sites 1 and 2 are reactive with different functional groups.

Many suitable heterobifunctional linkers are known in the art. See, e.g., S. S. Wong, Chemistry of Protein Conjugation and Crosslinking, CRC Press Inc., Boston 1991. In one aspect of the invention, Reactive Site No. 1 is a hydrazide, (i.e., a group of the formula

In one aspect of the invention, Reactive Site No. 2 is a thiol-accepting group. Suitable thiol-accepting groups include haloacetamide groups (i.e., groups of the formula

where X represents a halide) and maleimide groups (i.e., a group of the formula

In the heterobifunctional linker of the present invention, R¹⁶ is selected from arylene(lower alkylene), lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5.

In general, suitable heterobifunctional linkers include commercially available maleimido hydrazides (e.g., β-maleimido propionic acid hydrazide, ε-maleimidocaproic acid hydrazide, and SMCC hydrazide, available from Molecular Biosciences, Inc. Boulder Colo.). Alternatively, the heterobifunctional linker can be easily prepared from a maleimido acid, including polyoxoethylene-based maleimido acids (Frisch, B., et al., Bioconjugate Chem., 1996, 7, 180-186) according to known procedures (King, H. D., et al. Bioconjugate Chem., 1999, 10, 279-288). According to this procedure, maleimido acids are first converted to their N-hydroxysuccinimide esters, followed by reaction with tert-butylcarbazate. After purification and Boc removal the maleimido hydrazide are usually obtained in good yields. Haloacetamide hydrazide heterobifunctional linkers can be prepared from available intermediates, for example succinimidyl 3-(bromoacetamido)propionate, or [2-[2-(2-bromo-acetylamino)-ethoxy]-ethoxy]-acetic acid (Frisch, B., et al., Bioconjugate Chem., 1996, 7, 180-186) by the reaction with tert-butylcarbazate described above.

In another aspect of the invention, Reactive Site No. 2 is an amine-reactive group. Suitable amine reactive groups include activated esters such as N-hydroxysuccinimide esters, p-nitrophenyl esters, pentafluorophenyl esters, and other reactive functionalities such as isothiocyanates, isocyanates, anhydrides, acid chlorides, and sulfonyl chlorides.

As indicated previously, in a preferred embodiment, the synthesis of the drug should provide for the presence of a carbonyl group, or at least a group that can be readily converted to, or elaborated to form, a carbonyl group. A carbonyl group may be readily made part of, or added to, the pentapeptide drug by any of the following exemplary procedures:

(1) oxidation of a hydroxyl group of the drug to form a carbonyl;

(2) reaction of a hydroxyl group of the drug with a molecule that contains both a carbonyl or protected carbonyl and a hydroxyl-reactive group, e.g., a carboxylic acid or ester. For instance, the hydroxyl group of the drug may be reacted with a keto acid or a keto ester, so as to convert the hydroxyl group to an ester group, where the ester group is joined to the keto group of the keto acid/ester;

(3) introducing a carbonyl group to the drug during synthesis of the drug, e.g., during peptide synthesis using carbonyl-containing amino acids, amines or peptides.

For example, if the pentapeptide 10 includes a carbonyl group, then it can be reacted directly with a heterobifunctional linker containing a carbonyl-reactive end, e.g., a hydrazide group. If the pentapeptide 10 does not include a carbonyl group, then a carbonyl group may be introduced into the pentapeptide in order to impart hydrazide reactivity to the pentapeptide. For instance, when R⁹ is either of

then the pentapeptide 10 can be subjected to oxidizing conditions, e.g., pyridinium chlorochromate (PCC)/pyridine (see, e.g., Synthesis, 1982, 245, 881, review), in order to provide the corresponding oxidized compounds wherein R⁹ is

respectively.

Alternatively, if the pentapeptide 10 does not contain a carbonyl group but does contain a hydroxyl group, then this hydroxyl group can be elaborated to provide a carbonyl group. One way to provide carbonyl functionality from hydroxyl functionality is to react the hydroxyl group with a keto acid. The chemical literature describes many keto acids, and some keto acids are also available from commercial sources. Most keto acids, although not α and β aliphatic keto acids, can be readily condensed with a hydroxyl group using DCC/DMAP chemistry to provide keto esters 11 (Larock, R. C., Comprehensive Organic Transformations, Wiley-VCH, 1999, p. 1937). Exemplary keto acids include, without limitation: levulinic acid, 4-acetylbutyric acid, 7-oxooctanoic acid, 4-acetylbenzoic acid, 4-carboxyphenylacetone, N-methyl-4-acetyl-3,5-dimethyl-2-pyrrole carboxylic acid, 3-benzoyl-2-pyridinecarboxylic acid, and 2-acetylthiazol-4-carboxylic acid.

The use of an α-keto acid to provide a carbonyl group requires initial protection of the carbonyl group as, for example, a dimethyl ketal. Dimethyl ketals can be readily obtained by treatment of keto acids with an excess of dimethylorthoformate in the presence of concentrated sulfuric acid as described, for example, in LaMattina, J. L., Muse, D. E. J. Org. Chem. 1987, 52, 3479-3481. After protection, standard DCC/DMAP-mediated condensation of the carboxyl group of the keto acid with the hydroxyl group of the pentapeptide can be used to form the ester bond. After aqueous work-up and low vacuum drying, this material may be used without further purification. Once the ester bond is formed, the dimethyl ketal can be hydrolyzed under mild acidic conditions to give the desired α-keto ester 11. Exemplary α-keto acids include, without limitation: pyruvic acid, 2-ketobutyric acid, 2-thiophenglyoxylic acid, and benzoylformic acid.

As illustrated in Scheme 4, preparation of β-keto esters of pentapeptides may be performed via DMAP-promoted transesterification of ethyl β-keto esters (see, e.g., Otera, J. Chem. Rev., 1993, 93, 1449-1470). Commercially available β-keto esters that may be used in this transformation include, without limitation, ethyl acetoacetate, ethyl β-oxo-3-furanpropionate, and ethyl 3-(1-adamantyl)-3-oxopropionate. For instance, when R⁹ is selected from

the hydroxyl group of R⁹ may be reacted with a ketoacid to form an ester linkage and the desired carbonyl group. This approach to preparing carbonyl-containing drugs is shown in Scheme 4.

Once formed, the keto esters of pentapeptides can be isolated from the reaction mixture and purified by, for example, flash silica gel chromatography, and/or reversed phase high performance liquid chromatography.

Instead of utilizing an ester linkage to join the reactive carbonyl group (which may be reacted with a heterobifunctional linker) to the remainder of the drug, the reactive carbonyl group may be joined to the remainder of the drug via an ether linkage. That is, instead of providing a compound of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R⁹ is selected from

and R¹⁰ is

(i.e., an ester linkage in addition to R¹⁴ is used to link a reactive carbonyl group to the remainder of the molecule), the present invention also provides compounds wherein R¹⁰ is

(i.e., an ether linkage in addition to R¹⁴ is used to link a reactive carbonyl group to the remainder of the molecule) and, in either case (ether or ester linkage to R¹⁴) R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; and R¹⁵ is selected from hydrogen, lower alkyl and aryl.

The preparation of pentapeptides having an ether linkage to R¹⁴ is readily accomplished using amino alcohols (analogs of amino acids) which in turn are available from many commercial suppliers and are described in the chemical literature. The amino alcohol having a protected amino group (of general formula (protecting group)HN—R—OH where R is an organic moiety), may be reacted with an alcohol of the formula HO—R¹⁴—C(═O)—R¹⁵, for example via Mitsunobu reaction when R¹⁴=aryl, in the presence of PPh₃, DIAD and a suitable solvent, e.g., dioxane, to provide the corresponding amino ether (of the formula (protecting group)HN—R—O—R¹⁴—C(═O)—R¹⁵). Upon deprotection of the amino group, this amino group can be reacted with a carboxylic acid or a synthon thereof, to provide a peptide (amide) group as a step toward the preparation of a pentapeptide. This chemistry is illustrated in FIGS. 16 and 17, where an exemplary and preferred amino alcohol is compound 54.

After reaction with a heterobifunctional linker, the resulting product is a pentapeptide drug conjugated to a linker moiety, where the linker moiety is a reactive linker moiety. In other words, the pentapeptide-linker conjugate contains a group that is reactive with functionality present on the ligand.

Thus, in one aspect, the present invention provides a pentapeptide-linker conjugate of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety.

In another aspect, the present invention provides a pentapeptide-linker conjugate of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety.

In yet another aspect, the present invention provides a pentapeptide-linker conjugate of the formula

and pharmaceutically acceptable salts and solvates thereof wherein, independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocycle when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; and R²⁰ is a reactive linker group having a reactive site that allows R²⁰ to be reacted with a targeting moiety.

As shown in Scheme 5, hydrazone bond formation between the heterobifunctional linker and the carbonyl-containing pentapeptide drug, followed by treatment with the ligand, will provide compounds of formula I. The reaction of the drug with the heterobifunctional linker may be performed in a suitable solvent, e.g., anhydrous MeOH or DMSO, preferably in the presence of a small amount of acid, e.g., 0.01% AcOH or TFA at room temperature. The relative amounts of hydrazide to be applied and the time required for complete, or near complete reaction depends on the nature of the carbonyl group. Aromatic and α-carbonyl groups generally require higher excess of hydrazide and longer reaction time. The procedures of King, H. D., et al. Bioconjugate Chem., 1999, 10, 279-288, as used to prepare Doxorubicin-mAb conjugates, may be utilized to prepare the conjugates of the present invention. Stable hydrazones may be purified by a reversed phase HPLC. Hydrazone stability typically depends on the nature of corresponding keto esters.

The ligand is preferably a targeting ligand that directs the conjugate to a desired location in the animal subject. A preferred targeting ligand is an antibody, e.g., a chimeric or humanized antibody, or a fragment thereof.

Polyclonal antibodies which may be used in the compositions and methods of the invention are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest. For example, for the production of polyclonal antibodies, various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies which may be used in the compositions and methods of the invention are homogeneous populations of antibodies to a particular antigen (e.g., a cancer cell antigen, a viral antigen, a microbial antigen). A monoclonal antibody to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the more recent human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.

The monoclonal antibodies which may be used in the compositions and methods of the invention include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

The invention further provides for the use of bispecific antibodies. Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have differentspecificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low.

Similar procedures are disclosed in PCT Publication No. WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991, EMBO J. 10:3655-3659.

According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in PCT Publication No. WO 94/04690 published Mar. 3, 1994, which is incorporated herein by reference in its entirety.

For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210.

The invention provides for the use of functionally active fragments, derivatives or analogs of antibodies which immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens. Functionally active means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in a preferred embodiment the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay)

Other embodiments of the invention include fragments of the antibodies suitable for use in the invention such as, but not limited to, F(ab′)2 fragments, which contain the variable region, the light chain constant region and the CH1 domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. The invention also provides heavy chain and light chain dimers of the antibodies suitable for use in the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody of interest.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of which is incorporated herein by reference n its entirety.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of interest. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806; each of which is incorporated herein by reference in its entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).

The antibodies suitable for use in the invention include antibodies with modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, the antibodies suitable for use in the invention include antibodies with modifications in amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., PCT Publication No. WO 97/34631, which is incorporated herein by reference in its entirety).

Antibodies immunospecific for a cancer cell antigen can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.

In a specific embodiment, known antibodies for the treatment or prevention of cancer are used in accordance with the compositions and methods of the invention. Examples of antibodies available for the treatment of cancer include, but are not limited to, Herceptin® (Trastuzumab; Genentech, Calif.) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer (Stebbing, J., Copson, E., and O'Reilly, S. “Herceptin (trastuzamab) in advanced breast cancer” Cancer Treat. Rev. 26, 287-90, 2000); Rituxan® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG_(2a) antibody for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc., NY) which is murine IgG antibody for the treatment of lung cancer; IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgG antibody for the treatment of head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite, Mass.) which is a humanized IgG₁ antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart M195 (Protein Design Labs, Inc., CA) which is a humanized IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is a humanized antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA) which is a murine antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; anti-VEGF (Genentech, Inc., CA) which is humanized antibody for the treatment of lung and colorectal cancers; CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer; IMC-1C11 (ImClone Systems, NJ) which is an anti-KDR chimeric antibody for the treatment of colorectal cancer, lung cancers, and melanoma; and Cetuximab (ImClone, NJ) which is an anti-EGFR chimeric antibody for the treatment of epidermal growth factor positive cancers.

Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), MUC1 (carcinomas), placental alkaline phosphatase (carcinomas), prostate sepcific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 and MAGE 3 (carcinomas), anti-transferrin receptor (carcinomas), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), and CD40 (lymphoma). Some specific useful antibodies include BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellstrom, I., Hellstrom, K. E., “Cure of Xenografted Human Carcinomas by BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64 (Trail, PA, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J., Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza, A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R. S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom, K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability, Potency, and Efficacy of Carcinoma-reactive BR64-Doxorubicin Immunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against the CD 40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L., Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and in vivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res. 2000, 60, 3225-3231), and nmAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30 on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906, 1993). Many other internalizing antibodies that bind to tumor associated antigens can be used in this invention, and have been reviewed (Franke, A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy of acute myeloid leukenia: a review” Cancer Biother Radiopharm. 2000, 15, 459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: a coming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

Antibodies that are useful in this invention for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti Phospholipid Antibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; Anti SCL-70; Anti-Jo; Anti-UIRNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody.

In a specific embodiment, known antibodies for the treatment or prevention of viral or microbial infection are used in accordance with the compositions and methods of the invention. Examples of antibodies available for the treatment of viral infection or microbial infection include, but are not limited to, Synagis® (MedImmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of patients with RSV infection; PRO542 (Progenics) which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a human antibody for the treatment of hepatitis B virus; Protovir (Protein Design Labs, Inc., CA) which is a humanized IgG₁ antibody for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.

Other antibodies useful in the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).

Other antibodies useful in this invention for treatment of viral disease include antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picomaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis β virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.

The antibodies suitable for use in the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

Recombinant expression of the antibodies suitable for use in the invention, or fragment, derivative or analog thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody may be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

If an antibody that specifically recognizes a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an inmuunoglobulin), antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid sequence encoding at least the variable domain of the antibody is obtained, it may be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., PCT Publication No. WO 86/05807; PCT Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464). Vectors containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.

Once a nucleic acid sequence encoding an antibody of the invention has been obtained, the vector for the production of the antibody may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

The host cells used to express the recombinant antibody of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to express the immunoglobulin molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of the antibody may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the immunoglobulin molecule of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free gluta-thione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express an antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

The expression levels of an antibody can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Internalizing monoclonal antibodies, e.g., BR96, AC 10, herceptin, and S2C6 are preferred antibodies of the present invention. The ligand is suitably joined to the linker through a free thiol group or a free amino group on the ligand. For instance, when the ligand is an antibody, the free thiol group may be generated by reduction of a disulfide group present in the antibody with dithiothreitol as previously described (Trail, P. A. et al., Science 1993, 261, 212-215, and Firestone, R. A. Willner, D., Hofstead, S. J., King, H. D., Kaneko, T, Braslawsky, G. R., Greenfield, R. S., Trail, P. A., Lasch, S. J., Henderson, A. J., Casazza, A. M., Hellström, I., and Hellström, K. E., “Synthesis and antitumor activity of the immunoconjugate BR96-Dox” J. Controlled Rel. 1996, 39, 251-259). Alternatively, a lysine of the antibody may be reacted with iminothiolane hydrochloride (Traut's reagent) to introduce free thiol groups. Alternatively, free amino groups of the antibody may be reacted directly with linker-drug bearing suitable functionality, e.g., activated ester, isothiocyanate, isocyanate, anhydride, or acyl chloride functionalities. The drug-linker-ligand conjugate of the invention retains both specificity and therapeutic drug activity for the treatment of cell population expressing specific antigen for the ligand. Stable in serum, the hydrazone bond of the conjugate will be cleaved in acidic intracellular compartments releasing free highly-potent drug. This approach may be used to provide conjugates with high selectivity between target and non-target cell lines for example with up to about 0.1000 fold selectivity.

Scheme 5 illustrates the conjugation of the carbonyl-containing drug to a ligand through a heterobifunctional linker to form a compound of formula I.

wherein, in the compound of formula I, and independently at each location: R¹ is selected from hydrogen and lower alkyl; R² is selected from hydrogen and lower alkyl; R³ is lower alkyl; R⁴ is selected from lower alkyl, aryl, and —CH₂—C₅₋₇carbocyclic when R⁵ is selected from H and methyl, or R⁴ and R⁵ together form a carbocyclic group of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selected from hydrogen and lower alkyl and n is selected from 2, 3, 4, 5 and 6; R⁶ is selected from hydrogen and lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is selected from hydrogen and lower alkyl; R⁹ is selected from

R¹⁰ is selected from

R¹¹ is selected from hydrogen and lower alkyl; R¹² is selected from lower alkyl, halogen, and methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; R¹³ is

R¹⁴ is selected from a direct bond, arylene(lower alkylene), lower alkylene and arylene; R¹⁵ is selected from hydrogen, lower alkyl and aryl; R¹⁶ is selected from arylene(lower alkylene), lower alkylene, arylene, and —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is selected from

where Y=O or S. Compositions

In other aspects, the present invention provides pentapeptide drugs as described above, and prodrugs as also described above that are based on the pentapeptide drugs, in combination with a pharmaceutically acceptable carrier, excipient or diluent. For convenience, the pentapeptide drugs and the prodrugs of the invention will simply be referred to as compounds of the invention. Thus, the present invention provides a pharmaceutical or veterinary composition (hereinafter, simply referred to as a pharmaceutical composition) containing a compound of the invention as described above, in admixture with a pharmaceutically acceptable carrier. The invention further provides a composition, preferably a pharmaceutical composition, containing an effective amount of a compound as described above, in association with a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be in any form that allows for the composition to be administered to an animal subject. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably, the compositions are administered parenterally. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to an animal subject. Compositions that will be administered to a subject take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions should be pharmaceutically pure and non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the particular form of the active ingredient, the manner of administration, and the composition employed.

In general, the pharmaceutical composition includes an (where “a” and “an” refers here, and throughout this specification, to one or more) active compounds of the invention in admixture with one or more carriers. The carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) may be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.

When intended for oral administration, the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following adjuvants may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.

When the composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they are solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, tyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1% and about 80% of the weight of the composition. Preferred oral compositions contain between about 4% and about 50% of the compound of the invention. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01% to 2% by weight of active compound.

The present compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a present compound or composition. In certain embodiments, more than one compound or composition is administered to a subject. Methods of administration include but are not limited to oral administration and parenteral administration; parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous; intranasal, epidural, sublingual, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin. The preferred mode of administration is left to the discretion of the practitioner, and will depend in-part upon the site of the medical condition (such as the site of cancer or autoimmune disease).

In a preferred embodiment, the present compounds or compositions are administered parenterelly.

In a more preferred embodiment, the present compounds or compositions are administered intravenously.

In specific embodiments, it may be desirable to administer one or more compounds or compositions locally to the area in need of treatment. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre-neoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of an autoimmune disease.

In certain embodiments, it may be desirable to introduce one or more compounds or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the compounds or compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

In another embodiment, the compounds of the invention can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compounds or compositions can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled-release system can be placed in proximity of the target of the compounds or compositions, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer (Science 249:1527-1533 (1990)) may be used.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound of the invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a subject, the present compounds or compositions and pharmaceutically acceptable carriers are preferably sterile. Water is a preferred carrier when the compounds are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

In a preferred embodiment, the present compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, the compounds for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions may contain one or more optionally, agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profile as opposed to the spiked profiles of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade.

The amount of the compound or pharmaceutically acceptable salt or solvate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, dose ranges for a liquid composition intended for either parenteral or oral administration should contain an amount of a compound of the present invention such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a compound by weight in the composition. When intended for oral administration, this amount may be varied to a range from about 0.1% to about 80% of the weight of the composition. Preferred oral compositions contain from about 4% and about 50% of the compound by weight of the composition. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2%, preferrably from about 0.01% to about 2% of compound by weight of the parenteral dosage unit. For intravenous administration, the formulation preferably will be prepared so that the amount administered to the patient will be from about 1 to about 250 mg/kg of body weight of the desired conjugate. Preferably, the amount administered will be in the range of about 4 to about 25 mg/kg of body weight of the conjugate.

The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of a compound of the present invention of from about 0.1% to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

The composition may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule.

The pharmaceutical composition of the present invention may consist of gaseous dosage units, e.g., it may be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, spacers and the like, which together may form a kit. Preferred aerosols may be determined by one skilled in the art, without undue experimentation.

Whether in solid, liquid or gaseous form, the pharmaceutical composition of the present invention may contain one or more known pharmacological agents used in the treatment of cancer.

The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a compound of the invention with water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a compound of the invention so as to facilitate dissolution or homogeneous suspension of the active compound in the aqueous delivery system.

Biological Activity and Utility of Compounds

The present invention provides biologically-active compounds and pro-drugs (collectively, compounds, or compounds of the invention), methods of preparing compounds of the invention, pharmaceutical compositions comprising the compounds of the invention, and methods for treatment of cancers and other tumors in animal subjects. For instance, the invention provides compounds and compositions for use in a method for treating tumors wherein the animal subject is treated, in a pharmaceutically acceptable manner, with a pharmaceutically effective amount of a compound or composition of the present invention.

Treatment of Cancer

The compounds and compositions of the invention can be used in a variety of settings for the treatment of mammalian cancers. The mAb-pentapeptide conjugates can be used to deliver the cytotoxic drug to tumor cells. Without being bound by theory, once the antibody has bound to tumor associated antigens, it is taken up inside cells through receptor-mediated endocytosis into endosomes and lysosomes. These intracellular vesicles are acidic and can induce the hydrolysis of an acid-sensitive linker bond, e.g., a hydrazone bond, between the drugs and the mAbs. In addition, ester bonds can be cleaved by proteases and esterases, which are in abundance within lysosomes. The released drug is then free to migrate in the cytosol and induce cytotoxic activities. In an alternative embodiment, the drug is cleaved from the conjugate outside the cell and subsequently penetrates the cell.

The specificity of the mAb for a particular tumor type will dictate which tumors will be treated with the immunoconjugates. For example, BR96-pentapeptide conjugates may be used to treat antigen positive carcinomas including those of the lung, breast, colon, ovaries, and pancreas. Anti-CD30-pentapeptide and anti-CD40-pentapeptide conjugates may be used for treating hematologic malignancies.

Other particular types of cancers that may be treated with the compounds, compositions, and methods of the invention include, but are not limited to those disclosed in Table 1.

TABLE 1 Solid tumors, including but not limited to: fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer kidney cancer pancreatic cancer bone cancer breast cancer ovarian cancer prostate cancer esophogeal cancer stomach cancer oral cancer nasal cancer throat cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine cancer testicular cancer small cell lung carcinoma bladder carcinoma lung cancer epithelial carcinoma glioma glioblastoma multiforme astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma meningioma skin cancer melanoma neuroblastoma retinoblastoma blood-born cancers, including but not limited to: acute lymphoblastic leukemia “ALL” acute lymphoblastic B-cell leukemia acute lymphoblastic T-cell leukemia acute myeloblastic leukemia “AML” acute promyelocytic leukemia “APL” acute monoblastic leukemia acute erythroleukemic leukemia acute megakaryoblastic leukemia acute myelomonocytic leukemia acute nonlymphocyctic leukemia acute undifferentiated leukemia chronic myelocytic leukemia “CML” chronic lymphocytic leukemia “CLL” hairy cell leukemia multiple myeloma acute and chronic leukemias: lymphoblastic myelogenous lymphocytic myelocytic leukemias. Polycythemia vera Lymphomas: Hodgkin's disease non-Hodgkin's Lymphoma Multiple myeloma Waldenström's macroglobulinemia Heavy chain disease

The compounds and compositions of the invention may also be used as chemotherapeutics in the untargeted form. For example, the Pentapeptide drugs themselves, or the drug-linker conjugates minus the antibody moiety may be used against ovarian, CNS, renal, lung, colon, melanoma, and hematologic tumors.

Conjugation of highly potent drugs to monoclonal antibodies specific for tumor markers results in specific targeting, thus reducing general toxicity of these compounds. pH-Sensitive linkers provide stability of the conjugate in blood, yet are hydrolyzable in acidic intracellular compartments, liberating active drug.

Treatment of Autoimmune Diseases

The compounds pharmaceutically acceptable salts or solvates and compositions of the invention can also be used for the treatment of an autoimmune disease by killing or inhibiting the multiplication of cells which produce the auto-immune antibodies associated with said autoimmune disease.

Particular types of autoimmune diseases that may be treated with the compounds, compositions, and methods of the invention include, but are not limited to those disclosed in Table 2.

TABLE 2 Active Chronic Hepatitis Addison's Disease Ankylosing Spondylitis Anti-phosholipid Syndrome Arthritis Atopic Allergy Behcet's Disease Cardiomyopathy Celiac Disease Cogan's Syndrome Cold Agglutinin Disease Crohn's Disease Cushing's Syndrome Dermatomyositis Discoid Lupus Erythematosis Fibromyalgia Glomerulonephritis Goodpasture's Syndrome Graft v. Host Disease Grave's Disease Guillain-Barre Disease Hashimoto's Thyroiditis Idiopathic Adrenal Atrophy Idiopathic Pulmonary Fibritis IgA Nephropathy Inflammatory Bowel Diseases Insulin-dependent Diabetes Mellitus Juvenile Arthritis Lambert-Eaton Syndrome Lichen Planus Lupoid Hepatitis Lupus Lymphopenia Meniere's Disease Mixed Connective Tissue Disease Multiple Sclerosis Myasthenia Gravis Pernicious Anemia Polyglandular Syndromes Primary Agammaglobulinemia Primary Biliary Cirrhosis Psoriasis Psoriatic Arthritis Raynauds Phenomenon Reiter's Syndrome Rheumatoid Arthritis Schmidt's Syndrome Scleroderma Sjorgen's Syndrome Stiff-Man Syndrome Sympathetic Ophthalmia Systemic Lupus Erythematosis Takayasu's Arteritis Temporal Arteritis Thyrotoxicosis Type B Insulin Resistance Type I Diabetes Mellitus Ulcerative Colitis Uveitis Vitiligo Wegener's Granulomatosis Treatment of Cancer in Combination with other Treatment Modalities

Cancer or a neoplastic disease, including, but not limited to, a neoplasm, tumor, metastasis, or any disease or disorder characterized by uncontrolled cell growth, can be treated or prevented by administration of the compounds and compositions of the invention.

In other embodiments, a composition comprising one or more compounds of the invention or pharmaceutically acceptable salts or solvates thereof is administered along with radiation therapy and/or with one or a combination of chemotherapeutic agents, preferably with one or more chemotherapeutic agents with which treatment of the cancer has not been found to be refractory. The compounds of the invention or pharmaceutically acceptable salts or solvates thereof can be administered to a patient that has also undergone surgery as treatment for the cancer.

In another specific embodiment, the invention provides a method to treat or prevent cancer that has shown to be refractory to treatment with a chemotherapy and/or radiation therapy.

In a specific embodiment, a composition comprising one or more compounds of the invention or pharmaceutically acceptable salts or solvates thereof is administered concurrently with chemotherapy or radiation therapy. In another specific embodiment, chemotherapy or radiation therapy is administered prior or subsequent to administration of a present composition, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e.g., up to three months), subsequent to administration of a therapeutic of the invention.

The chemotherapy or radiation therapy administered concurrently with, or prior or subsequent to, the administration of a present composition can be accomplished by any method known in the art. The chemotherapeutic agents are preferably administered in a series of sessions, any one or a combination of the chemotherapeutic agents listed above can be administered. With respect to radiation therapy, any radiation therapy protocol can be used depending upon the type of cancer to be treated. For example, but not by way of limitation, x-ray radiation can be administered; in particular, high-energy megavoltage (radiation of greater that 1 MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-ray radiation can be used for skin cancers. Gamma-ray emitting radioisotopes, such: as radioactive isotopes of radium, cobalt and other elements, may also be administered to expose tissues to radiation.

Additionally, the invention provides methods of treatment of cancer or neoplastic disease with a present composition as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or may prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The subject being treated with the present compositions may, optionally, be treated with other cancer treatments such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.

The present compositions may also be used in an in vitro fashion, such as for the treatment of certain cancers, including, but not limited to leukemias and lymphomas, such treatment involving autologous stem cell transplants. This involves a multi-step process in which the patient's autologous hematopoietic stem cells are harvested and purged of all cancer cells, the patient's remaining bone marrow cell population is then eradicated via the administration of a high dose of a composition of the invention +/−high dose radiation therapy, the stem cell graft is infused back into the patient, and supportive care is provided while bone marrow function is restored and the patient recovers.

Combination Therapy

In certain embodiments of the present invention, the compounds and pharmaceutically acceptable salts or solvates thereof can be used in combination with at least one other therapeutic agent.

The compound(s) and the other therapeutic agent(s) can act additively or, more preferably, synergistically. In a preferred embodiment, a composition comprising a compound is administered concurrently with the administration of one or more additional therapeutic agent(s), which can be part of the same composition or in a different composition from that comprising the compound. In another embodiment, a composition comprising the compound is administered prior to or subsequent to administration of another therapeutic agent(s).

In certain embodiments, the compounds can be used in combination with one or more anticancer agents and pharmaceutically acceptable salts or solvates thereof. Suitable anticancer agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred embodiment, one or more of the compositions of the invention is used to treat or prevent cancer or neoplastic disease in combination with one or more chemotherapeutic or other anti-cancer agents including, but not limited to those presented in Table 3.

TABLE 3 Alkylating agents Nitrogen mustards: cyclophosphamide Ifosfamide trofosfamide Chlorambucil Nitrosoureas: carmustine (BCNU) Lomustine (CCNU) Alkylsulphonates busulfan Treosulfan Triazenes: Dacarbazine Platinum containing compounds: Cisplatin carboplatin Plant Alkaloids Vinca alkaloids: vincristine Vinblastine Vindesine Vinorelbine Taxoids: paclitaxel Docetaxol DNA Topoisomerase Inhibitors Epipodophyllins: etoposide Teniposide Topotecan 9-aminocamptothecin campto irinotecan crisnatol mitomycins: Mitomycin C Anti-metabolites Anti-folates: DHFR inhibitors: methotrexate Trimetrexate IMP dehydrogenase Inhibitors: mycophenolic acid Tiazofurin Ribavirin EICAR Ribonuclotide reductase Inhibitors: hydroxyurea deferoxamine Pyrimidine analogs: Uracil analogs 5-Fluorouracil Floxuridine Doxifluridine Ratitrexed Cytosine analogs cytarabine (ara C) Cytosine arabinoside fludarabine Purine analogs: mercaptopurine Thioguanine Hormonal therapies: Receptor antagonists: Anti-estrogens Tamoxifen Raloxifene megestrol LHRH agonists: goscrclin Leuprolide acetate Anti-androgens: flutamide bicalutamide Retinoids/Deltoids Vitamin D3 analogs: EB 1089 CB 1093 KH 1060 Photodyamic therapies: vertoporfm (BPD-MA) Phthalocyanine photosensitizer Pc4 Demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines: Interferon-α Interferon-γ Tumor necrosis factor Others: Isoprenylation inhibitors: Lovastatin Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell cycle inhibitors: staurosporine Actinomycins: Actinomycin D Dactinomycin Bleomycins: bleomycin A2 Bleomycin B2 Peplomycin Anthracyclines: daunorubicin Doxorubicin (adriamycin) Idarubicin Epirubicin Pirarubicin Zorubicin Mitoxantrone MDR inhibitors: verapamil Ca²⁺ATPase inhibitors: thapsigargin

In another preferred embodiment, the compounds can be used in combination with one or more immunosuppressant agents and pharmaceutically acceptable salts or solvates thereof to treat an autoimmune disease. In a preferred embodiment, the immunosuppressant agent includes, but is not limited to, cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus, tacrolimus, enanercept, prednisone and azathioprine.

In still another embodiment, the other therapeutic agent can also be an adjuvant to reduce any potential side effects of treatment, such as, for example, an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.

The present compounds and compositions may also be used alone or in combination with one or more antitumor agents and one or more immunosuppressant agents to simultaneously treat a combination of cancer and an autoimmune disease.

The free drugs of the invention may also be used as chemotherapeutics in the untargeted form. For example, the pentapeptides of the present invention may be used against ovarian, CNS, renal, lung, colon, melanoma, and hematologic tumors.

Conjugation of these highly potent drugs to monoclonal antibodies specific for tumor markers results in specific targeting, thus reducing general toxicity of these compounds. pH-Sensitive linkers should provide stability of the conjugate in blood, yet should be hydrolyzed in acidic intracellular compartments liberating active drug.

The following examples are provided by way of illustration and not limitation.

EXAMPLES

The chemistry described in Examples 1-4 is illustrated in the schemes shown in FIG. 1-3. The chemistry described in Examples 5 and 6 is illustrated in the scheme shown in FIG. 4. The chemistry described in Example 7 is illustrated in the scheme shown in FIG. 5. The chemistry described in Example 8 to prepare compound 36 is illustrated in the scheme shown in FIG. 6, while the conjugation of compound 36 to an antibody is illustrated in FIG. 10. The chemistry described in Example 9 to prepare compounds 38 is illustrated in the scheme shown in FIG. 7. Chemistries to make compounds 40a and 40b, as described in Examples 10 and 11, are shown in FIG. 8, while the conjugation of 40a to an antibody is illustrated in FIG. 11. The preparation of drug-linker-mab 42 as described in Example 12 is illustrated in the scheme shown in FIG. 9. The reaction of pentapeptide 47 with a heterobifunctional linker as described in Example 13 is illustrated in the scheme of FIG. 12, where this scheme also shows the formation of the drug-linker-antibody conjugate mAb-S-48. The scheme of FIG. 13 shows the preparation of the pentapeptide 49 as described in Example 14. The synthetic descriptions provided in Examples 15, 16, 17 and 18 are illustrated in the schemes provided in FIGS. 14, 15, 16 and 17, respectively.

Coupling Procedure A: Peptide Synthesis Using DEPC

To a cooled (ice bath) solution of [2S-[2R*(αS*,βS*)]]-1-[(1,1-dimethylethoxy) carbonyl]-β-methoxy-α-methyl-2-pyrrolidinepropanoic acid (t-Boc-dolaproine (20) 0.27 g, 0.94 mmol) in anhydrous CH₂Cl₂ (10 mL) is added an identified amine (1.03 mmol, 1.1 eq.) followed by triethylamine (0.40 mL, 2.8 mmol, 3.0 eq.) and DEPC (0.17 mL, 90%, 1.03 mmol, 1.1 eq.). The resulting solution is stirred under argon for 12 hours. The solvent is removed under reduced pressure at room temperature, and the residue is chromatographed (silica gel column using 4:1 hexanes-acetone as eluent). After the evaporation of solvent from the fractions selected according to TLC analysis, the residue is dried under vacuum overnight to afford the amide.

Coupling Procedure B: Peptide Synthesis Using PyBrop

The amino acid (1.0 eq.) containing a carboxylprotecting group, if necessary, was dissolved in anhydrous CH₂Cl₂ followed by the addition of diisopropylethylamine (1.5 eq.). Fmoc-, Z-, or dimethyl amino acid/pepide (1.1 eq.) was added as a solid in one portion and the dissolved mixture had added PyBrop (1.2 eq.). The reaction was monitored by TLC or HPLC.

General Procedure C: Pentapeptide Synthesis

A solution of dipeptide and tripeptide (1 eq. each) in CH₂Cl₂ (10 mL) and trufluoroacetic acid (5 mL) is stirred (ice bath under a N₂ atmosphere) for two hours. The reaction may be monitored by TLC or, preferably, HPLC. The solvent is removed under reduced pressure and the residue dissolved in toluene. Solvent is again removed in vacuo and the process repeated. The residue is dried under high vacuum for 12 h and then dissolved in dry CH₂Cl₂ followed by the addition of diisopropylethylamine (1.5 eq.). Depending on the residues, the peptides may be coupled using either PyBrop or DEPC (1.2 eq.). The reaction mixture is monitored by either TLC or HPLC.

General procedure D: Z-Removal via Hydroienolysis

Using a large, heavy-walled flask, a solution of Z-protected amino acid or peptide was dissolved in ethanol. 10% palladium on carbon was added (1% w/w peptide) and the mixture was introduced to H₂. Reaction progress was monitored by HPLC and typically found to be complete within 1-2 h. The flask contents are filtered through a pre-washed pad of celite and then the celite is washed with methanol. The eluent solution is evaporated to an oil, dissolved in toluene, and re-evaporated.

General Procedure E: Fmoc-Removal Using Diethylamine

The Fmoc-containing compound is dissolved in CH₂Cl₂ to which an equal amount of diethylamine is added. Reaction progress is monitored by TLC (or HPLC) and is usually complete within 2 h. Solvents are removed in vacuo and the residue taken up in toluene and concentrated again. The residue is dried under high vacuum for at least 1 h.

General Procedure F: Hydrazone Formation

Hydrazone formation is performed in anhydrous MeOH, 0.01% AcOH (typically 1 mL per 10 mg of a drug) at room temperature. Time of the reaction (6 h-5 days) and an amount of a hydrazide (2-30 eq.) may be varied depending on the specific ketone. The reaction progress is monitored by C₁₈ RP-HPLC. Typically, a newly formed acylhydrazone has lower retention time compared to the parent ketone. After the reaction is complete, DMSO (1-2 mL) is added to the reaction mixture and methanol is removed under reduced pressure. The residue is directly loaded onto a C₁₈ RP column for preparative HPLC purification (Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 100 mM TEAA buffer, pH 7.0, from 10 to 95% at flow rate 4 mL/min). The appropriate fractions are concentrated under reduced pressure, co-evaporated with acetonitrile (4×25 mL), and finally dried in deep vacuum for 2 days.

Example 1 Preparation of Compound 29a

Preparation of Compound 14a

Compound 14a was prepared by reacting compounds 12 and 13a according to coupling procedure B. After concentration of the reaction mixture, the residue was directly injected onto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using an isocratic run of 25% aqueous MeCN at 20 mL/min) in order to remove the unwanted diastereomer. The pure fractions were concentrated to give the product as a clear oil. Yield 14a: 0.67 g (55%); ES-MS m/z 493.4 [M+H]⁺; UV λ_(max) 215,256 nm.

Preparation of Compound 15a

Compound 14a was treated according to deprotection procedure D to provide compound 15a.

Preparation of Compound 18a

Compounds 15a and 16 were coupled and characterized as described in Pettit et. al. J. Chem. Soc. Perk I, 1996, 859.

Preparation of [(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidine-1-carboxylic acid-tert-butyl ester (25)

t-Boc-dolaproine 20 and (1S,2R)-norephedrine 21 were combined in the presence of DEPC and triethylamine according to General procedure A (see also U.S. Pat. No. 5,635,483 to Pettit et al.) to provide compound 25.

Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (29a, Auristatin E)

Compound 18a and 25 were combined in the presence of trifluoroacetic acid in methylene chloride (1:1), followed by treatment with DEPC and triethylamine, to provide compound 29a using procedure C. The synthesis of this compound is also reported in U.S. Pat. No. 5,635,483, and Pettit et al., Anti-Cancer Drug Des. 1998, 243.

Example 2 Preparation of Compound 29b

Preparation of Compound 14b

According to coupling procedure B, this peptide was prepared using compounds 12 and 13b. After concentration of the reaction mixture, the residue was purified by C₁₈ preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using an isocratic run of 75% aqueous MeCN at 20 mL/min) in order to remove the unwanted diastereomer. The pure fractions were concentrated to a clear oil. Yield 14b: 0.67 g (55%); R_(f) 0.32 (4:1 hexanes-acetone); UV λ_(max) 215, 256 nm; ¹H NMR (CDCl₃) δ 7.14-7.40(5 H, m), 6.19 (1 H, d, J=9.3 Hz), 5.09 (2 H, s), 4.53 (1 H, dd, J=6.6, 9.3 Hz), 3.34 (3 H, s), 2.97 (3 H, s), 2.25-2.50 (2 H, m), 1.50-1.78 (3 H, m), 1.46 (9 H, s), 0.99 (3 H, d, J=6.9 Hz), 0.96 (3 H, d, J=6.9 Hz), 0.89 (3 H, t, J=6.9 Hz), 0.83 (3 H, t, J=7.2 Hz).

Preparation of Compound 15b

Compound 14b was treated according to deprotection procedure D to provide compound 15b.

Preparation of Compound 18b

Compounds 15b and 16 were combined using procedure A to provide compound 18b: Yield 18b: 0.19 g (82%); ES-MS m/z 500.5 [M+H]⁺; R_(f) 0.12 (4:1 hexanes-acetone); UV λ_(max) 215 run.

Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-isoleucinamide, (29b)

Following general procedure C, compounds 18b and 25 were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and triethylamine. The reaction mixture was purified by preparative:HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 100 mM aqueous triethylammonium carbonate from 10 to 100% in 40 min followed by 20 min at 0% buffer, at flow rate 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 29b: 85 mg (60%); ES-MS m/z 746.5 [M+H]⁺; UV λ_(max) 215 nm.

Example 3 Preparation of Compound 31a

Preparation of Compound 19a

Compounds 15a and 17 were combined using procedure A to provide compound 19a: 107 mg (50%); ES-MS m/z 694.7 [M+H]⁺; R_(f) 0.64 (1:1 hexanes-EtOAc); UV λ_(max) 215, 265 nm.

Preparation of Compound 30a

Following general procedure C, compounds 19a and 25 (prepared as described in Example 1) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and triethylamine. The reaction mixture was monitored by HPLC and after 4 h water was added and the layers separated. After drying (MgSO₄), the contents were filtered and solvent removed to give compound 30a that was used in the next step without further purification. Yield 30a: 127 mg (91%); ES-MS m/z 940.9 [M+H]⁺.

Preparation of N-methyl-L-valyl-N-[(1S,2R)-4-[(2g)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (31 a)

The Fmoc-protected peptide was treated with diethylamine according to deprotection procedure E. A complete reaction was observed by HPLC after 12 h. The reaction mixture was concentrated to an oil and purified by preparative-HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The desired fractions were pooled and concentrated to give an off-white solid. Yield 31a: 37 mg (38%); ES-MS m/z 718.7. [M+H]⁺; UV λ_(max) 215 nm.

Example 4 Preparation of Compound 31b

Preparation of Compound 19b

Compounds 15b and 17 were combined using procedure A to provide compound 19b. Yield 19b: 0.50 g (73%); UV λ_(max) 215, 265 nm.

Preparation of Compound 30b

Following general procedure C, compounds 19b and 25 (prepared as described in Example 1) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and triethylamine. The reaction mixture was purified by preparative-HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 30b: 64 mg (53%); ES-MS m/z 955.2 [M+H]⁺; UV λ_(max) 215, 265 nm.

Preparation of N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide, (31 b)

The Fmoc-protected peptide 30b was treated with diethylamine according to deprotection procedure E. A complete reaction was observed by HPLC after 2 h. The reaction mixture was concentrated to an oil. Excess ether was added resulting in a white precipitate and the contents were cooled to 0° C. for 3 h. The product was filtered and the solid dried under high vacuum. Yield 31b: 47 mg (95%); ES-MS m/z 732.8 [M+H]⁺; UV λ_(max) 215 mm.

Example 5 Preparation of Compound 32

Preparation of [(2S)-2-[(1R,2R)-3-[[(1S)-hydroxymethyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidine-1-carboxylic acid-tert-butyl ester (26)

The dipeptide 26 was synthesized from t-Boc-dolaproine (20) and (S)-(−)-2-amino-3-phenyl-1-propanol (L-phenylalaninol, 22) according to General procedure A. Yield 26: 0.50 g (55%); ES-MS m/z 421.0 [M+H]⁺; R_(f) 0.24 (100% EtOAc); UV λ_(max) 215, 256 nm. ¹H NMR (CDCl₃) δ 7.14-7.40 (5 H, m), 6.19 (1 H, d, J=7.8 Hz), 4.11-4.28 (1 H, m), 3.44 (3 H, s), 3.20-3.84 (5 H, m), 2.80-3.05 (2 H, m), 2.20-2.38 (2 H, m), 1.65-1.98 (4H, m), 1.48(9 H, s), 1.16 (3 H, d, J=5.7 Hz).

Preparation of Compound 32

Following general procedure C, compounds 26 (42 mg, 0.1 mmol, 1 eq.) and 18a (65 mg, 0.13 mmol, 1.3 eq.) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and diisopropylethylamine. The reaction mixture was purified by preparative-HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 32: 60 mg (80%); ES-MS m/z 732.2 [M+H]⁺; UV λ_(max) 215, 265 nm.

Example 6 Preparation of Compound 33

[(2S)-2-[(1R,2R)-3-[[(1R)-hydroxymethyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-pyrrolidine-1-carboxylic acid-tert-butyl ester (27)

The dipeptide 27 was synthesized from t-Boc-dolaproine (20) and (R)-(+)-2-amino-3-phenyl-1-propanol (D-phenylalaninol, 23) according to General procedure A. Yield 27: 0.53 g (58%); ES-MS m/z 421.0 [M+H]⁺; R_(f) 0.24 (100% EtOAc); UV λ_(max) 215, 256 nm. ¹H NMR (CDCl₃) δ 7.14-7.40 (5 H, m), 6.19 (0.5 H, d, J=5.4 Hz), 5.75 (0.5 H, d, J=5.4 Hz), 4.10-4.23 (1 H, m), 3.38 (3 H, s), 3.10-3.85 (4 H, m), 2.82-2.96 (2 H, m), 2.10-2.36(2 H, m), 1.66-1.87 (4 H, m), 1.47 (9 H, bs), 1.16 (1.5 H, d, J=6.6 Hz), 1.08(1.5 H, d, J=5.7 Hz).

Compound 33

Following general procedure C, compounds 27 (42 mg, 0.1 mmol, 1 eq.) and 18a (65 mg, 0.13 mmol, 1.3 eq.) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and diisopropylethylamine. The reaction mixture was purified by preparative-HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 25 to 70% in 5 min followed by 30 min at 70%, at a flow rate of 20 mL/min). The product was isolated as an off-white solid after concentration of the desired HPLC fractions. Yield 33: 34 mg (46%); ES-MS m/z 732.1 [M+H]⁺; UV λ_(max) 215, 265 nm.

Example 7 Preparation of Compound 34

Preparation of Compound 28

Dipeptide 28 is prepared by reacting Boc-dolaproine 20 and p-acetylphenethylamine (U.S. Pat. No. 3,445,518, 1969) according to coupling procedure B. After concentration of the reaction mixture, the residue is purified by reverse phase preparative-HPLC or via SiO₂ chromatography.

Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(4-acetylphenyl)ethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide. (34)

Dipeptide 28 and tripeptide 18a are combined in the presence of trifluoroacetic acid in methylene chloride, following a coupling reaction with DEPC and triethylamine as described in general procedure C. The reaction mixture is purified under usual preparative-HPLC measures as previously described for other peptide compounds.

Example 8 Preparation of Compound 36 Preparation of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R)-2-oxo-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide (35)

Pyridinium chlorochromate (PCC) (13.6 mg, 0.06 mmol, 4.5 eq.) was added to a solution of Auristatin E (29a) (10 mg, 0.014 mmol, 1 eq.) in CH₂Cl₂ (2 mL) and pyridine (50 μL). The reaction mixture was stirred at room temperature for 3 h. HPLC analysis of the reaction mixture showed complete conversion of the 10.2 min peak into a new 11.3 min peak with different UV spectrum (max 245 nm, shoulder 280 nm). The product was purified by flash chromatography on a silica gel column (120×12 mm) in a step gradient of MeOH in CH₂Cl₂ from 0 to 10%. After concentration in vacuum, the residue was triturated with hexane to give white solid. Yield 35: 6.4 mg (64%); R_(f) 0.3 (CH₂Cl₂/MeOH, 10/1); UV λ_(max) 215, 245 nm. HRMS m/z: found 730.5119 [M+H]⁺. C₄₀H₆₈N₅O₇ requires 730.5108.

Preparation of Hydrazone 36

Compound 36 was prepared from compound 35 by reaction with 30 eq. of maleimidocaproylhydrazide for 3 days according to General Procedure F. Yield 36: 2.4 mg (43%) of colorless glass; UV λ_(max) 215, 240 (shoulder) nm. HRMS m/z: found 937.6131 [M+H]⁺. C₅₀H₈₁N₈O₉ requires 937.6127.

Example 9 Preparation of Compound 38

Preparation of Levulinic Ester of Auristatin E (37)

Levulinic acid (2.5 μL, 0.025 mmol, 5 eq.) was added to a solution of auristatin E (29a, 3.6 mg, 0.005 mmol, 1 eq.) in anhydrous CH₂Cl₂ (1 ml), followed by DCC (5 mg, 0.025 mmol, 5 eq.) and DMAP (1 mg, cat). After reacting for overnight at room temperature, analysis by C₁₈ RP-HPLC revealed formation of a new, more hydrophobic product. Precipitated DCU was filtered off. The resulting keto ester was isolated using preparative chromatography on silica gel in a step gradient of MeOH in CH₂Cl₂ from 0 to 10%. Yield 37: 3.3 mg (80.5%) of colorless glass; R_(f) 0.35 (CH₂Cl₂/MeOH, 10/1); ES-MS m/z 830 [M+H]⁺; UV λ_(max) 215 nm.

Preparation of Hydrazone 38

Compound 38 was prepared from compound 37 and maleimidocaproylhydrazide (2 eq.) according to General Procedure F, reaction time—6 h. Yield 38: 1.3 mg (46%) of white solid, ES-MS m/z 1037 [M+H]⁺. UV λ_(max) 215 nm.

Example 10 Preparation of Compound 40a

Preparation of 4-acetyl-benzoic Ester of Auristatin E (39a)

4-Acetylbenzoic acid (23.5 mg, 0.14 mmol, 2 eq.) was added to a solution of auristatin E (29a, 50 mg, 0.07 mmol, 1 eq.) in anhydrous CH₂Cl₂ (5 ml), followed by DCC (30 mg, 0.14 mmol, 2 eq.) and DMAP (5 mg, cat). After reacting overnight at room temperature, analysis by C₁₈ RP-HPLC revealed formation of a new, more hydrophobic product. Precipitated DCU was filtered off. The resulting keto ester was isolated using preparative chromatography on silica gel in a step gradient of MeOH in CH₂Cl₂ from 0 to 10%. The product was eluted with 5% MeOH in CH₂Cl₂. Yield 39a: 57 mg (90%) of white solid; R_(f) 0.43 (CH₂Cl₂/MeOH, 10/1); UV λ_(max) 215, 250 nm. HRMS m/z: found 878.5669 [M+H]⁺. C₄₉H₇₆N₅O₉ requires 878.5643. Anal. calcd for C₄₉H₇₅N₅O₉ ×H₂O: C, 65.67; H, 8.66; N, 7.81. Found: C, 66.05; H, 8.61; N, 7.80.

Preparation of Hydrazone 40a

Compound 40a was prepared from compound 39a and maleimidocaproylhydrazide (3 eq.) according to General Procedure F, reaction time—12 h. Yield 40a: 3 mg (65%) of solid; UV λ_(max) 215, 295 nm. HRMS m/z: found 1085.6665 [M+H]⁺. C₅₉H₈₉N₈O₁₁ requires 1085.6651. Anal. calcd for C₅₉H₈₈N₈O₁₁×H₂O: C, 64.22; H, 8.22; N, 10.16. Found: C, 64.27; H, 8.21; N, 9.92.

Example 11 Preparation of Compound 40b Preparation of 4-acetyl-benzoic ester of N,N-dimethyl-L-valyl-N-[(1S,2R)-4-[(2s)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-isoleucinamide. (39b)

Compound 39b was prepared from compound 29b by reaction with 4-acetobenzoic acid in the presence of DCC, DMAP in CH₂Cl₂ as described in the Example 10. Yield 39b: 10.7 mg (80%) of white solid; R_(f) 0.37 (CH₂Cl₂/MeOH, 10/1); UV λ_(max) 215, 250 nm. HRMS m/z: found 892.5775 [M+H]⁺. CsoH₇₈N₅O₉ requires 892.5800.

Preparation of Hydrazone (40b)

Compound 40b was prepared from compound 39b and maleimidocaproylhydrazide (5 eq.) according to General Procedure F, reaction time—18 h. Yield 40b: 4.8 mg (50%) of solid; ES-MS m/z: 1099 [M+H]⁺; UV λ_(max) 215, 295 nm.

Example 12 Preparation of Compound 42

Preparation of Compound 42

To a solution of 39a (5 mg, 0.0056 mmol) in DMSO (100 μL) anhydrous MeOH (0.9 mL) was added, followed by 1% AcOH/MeOH (10 μL). 3-Bromoacetamido)propyonyl hydrazide (41) (0.025 mmol, 4.5 eq.) was added to the solution. The reaction was left at room temperature for 24 h. C₁₈-RP HPLC analysis revealed the formation of a new compound with a lower retention time. DMSO (1 mL) was added to the reaction mixture and methanol was removed under reduced pressure. The residue was directly loaded onto a C₁₈-RP column for preparative HPLC purification (Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 100 mM TEAA buffer, pH 7.0, from 10 to 95% at flow rate 4 mL/min). The appropriate fractions are concentrated under reduced pressure, co-evaporated with acetonitrile (4×25 mL), and finally dried in deep vacuum. Yield 42: 4.0 mg (66%) of solid; ES-MS m/z: 1084 [M+H]⁺, 1086; UV λ_(max) 215, 295 nm.

Example 13 Preparation of Compound 48

Compound 47

Compound 47, shown below, was prepared from compound 32 (8.8 mg, 0.012 mmol) by reaction with 4-acetobenzoic acid in the presence of DCC, DMAP in CH₂Cl₂ as described in the Example 10. Yield 47: 7.3 mg (70%) of white solid; R_(f) 0.4 (CH₂Cl₂/MeOH, 10/1); ES-MS m/z 878.9 [M+H]⁺; UV λ_(max) 215, 250 nm.

Preparation of Hydrazone 48

Compound 48 was prepared from compound 47 (6.4 mg, 0.007 mmol) and maleimidocaproylhydrazide (4 eq.) according to General Procedure F, reaction time—4 h. Yield 48: 4.3 mg (57%) of colorless glass; ES-MS m/z: 1085.7 [M+H]⁺; UV λ_(max) 215, 294 mm.

Example 14 Preparation of Compound 49

Compound 49 (structure shown above) was prepared from compound 33 (8.0 mg, 0.01 mmol) by reaction with 4-acetobenzoic acid in the presence of DCC, DMAP in CH₂Cl₂ as described in the Example 10. Yield 49: 8.1 mg (92%) of white solid; R_(f) 0.37 (CH₂Cl₂/MeOH, 10/1); UV λ_(max) 215, 250 nm. HRMS m/z: found 878.5653 [M+H]⁺. C₄₉H₇₆N₅O₉ requires 878.5643.

Example 15 Preparation of Compound 51

Preparation of 5-benzoylvaleric Ester of Auristatin E (50)

Compound 50 (structure shown below) was prepared from auristatin E (29a, 10 mg, 0.014 mmol) by reaction with 5-benzoylvaleric acid in the presence of DCC, DMAP in CH₂Cl₂ as described in the Example 10. Yield 50: 10 mg (79%) of pale yellow oil; R_(f) 0.34 (CH₂Cl₂/MeOH, 10/1); UV λ_(max) 215 nm. HRMS m/z: found 920.6139 [M+H]⁺. C₅₂H₈₂N₅O₉ requires 920.6113.

Preparation of Hydrazone 51

Compound 51 was prepared from compound 50(5 mg, 0.0054 mmol) and maleimidocaproylhydrazide (5 eq.) according to General Procedure F, reaction time—12 h. Yield 51: 3.5 mg (57%) of white solid; UV λ_(max) 215, 280 nm. HRMS m/z: found 1127.7132 [M+H]⁺. C₆₂H₉₅N₈O₁₁ requires 1127.7120.

Example 16 Preparation of Compound 53

Preparation of Acetoacetic Ester of Auristatin E (52)

A solution of auristatin E (29a, 10 mg, 0.014 mmol, 1 eq.), ethyl acetoacetate (9 μL, 0.07 mmol, 5 eq.), and DMAP (3.5 mg, 0.029 mmol, 2 eq.) in anhydrous toluene (5 mL) was refluxed for 10 h. The reaction mixture was concentrated under reduced pressure to dryness. The residue was re-dissolved in 2 mL of CH₂Cl₂ and the product was purified by silica gel flash chromatography in 10% MeOH in CH₂Cl₂. Yield 52 (structure shown below): 7 mg (60%) of colorless glass; R_(f) 0.4 (CH₂Cl₂/MeOH, 10/1); ES-MS m/z: 816.9 [M+H]⁺; UV λ_(max) 215 nm.

Preparation of Hydrazone 53

Compound 53 was prepared from compound 52 and maleimidocaproylhydrazide (10 eq.) according to General Procedure F, reaction time—12 h. Hydrazone 53 was used for kinetic studies without isolation. UV λ_(max) 215 nm.

Example 17 Preparation of Compound 57

Preparation of Boc-Phenylalamino]-p-acetylphenoxy ether (56)

A solution (S)-(−)-Boc-phenylalaminol (54, 0.50 g, 2.0 mmol, 1.0 eq.), 4-hydroxyacetophenone (55, 0.30 g, 2.2 mmol, 1.1 eq.), and triphenylphosphine (0.80 g, 3.0 mmol, 1.5 mmol), in anhydrous dioxane (20 mL) was cooled to 0° C. Dropwise addition of diisopropylazodicarboxylate (0.62 mL, 3.0 mmol, 1.5 eq.) was performed over a 2 min period and the reaction was monitored by reverse phase-HPLC. After 24 h, solvent was removed in vacuo and the residue purified by preparative-HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 20 mL/min). The desired fractions were concentrated to give the product as a white solid. Yield 56: 0.54 g (74%); ES-MS m/z 370.2 [M+H]⁺; UV λ_(max) 215, 275 nm. ¹H NMR (CDCl₃) δ 7.96 (2 H, d, J=6.9 Hz), 7.18-7.35 (5H, m), 6.94 (2 H, d, J=8.4 Hz), 4.92 (1 H, bd, J=8.1 Hz), 4.14-4.28 (1 H, m), 3.91-4.02 (2 H, m), 2.99-3.04 (2 H, m), 2.58 (3 H, s), 1.45 (9 H, s).

Preparation of Dipeptide 57

A solution of 56 (70 mg, 0.19 mmol, 1.0 eq.) in 10 mL CH₂Cl₂-TFA (2:1) stood over a N₂ atmosphere for 2 h. HPLC indicated a complete reaction. The reaction mixture was concentrated to an oil that was taken up in a minimum amount of dichloromethane and precipitated with hexanes. The white solid was collected and dried under high vacuum for 20 h. The free amine and t-Boc-dolaproine (20) were combined in the presence of DEPC (1.5 eq.) and triethylamine (3.0 eq.) according to General procedure A. After 24 h, solvent was removed in vacuo and the residue purified by preparative-HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of 20 mL/min). The desired fractions were concentrated to give the product as a white solid. Yield 57: 52 mg (51%); ES-MS m/z 538.9 [M+H]⁺; UV λ_(max) 215, 275 nm. ¹H NMR (CDCl₃) δ 7.92 (2 H, d, J=7.2 Hz), 7.18-7.33 (5 H, m), 6.94 (2 H, d, J=7.5 Hz), 6.69 (½ H, bd, J=7.2 Hz), 5.98 (½ H, bd, J=7.5 Hz), 4.46-4.57 (1 H, m), 3.96-4.08 (2 H, m), 3.48-3.88 (3 H, m), 3.38 (3 H, s), 2.92-3.26 (3 H, m), 2.56 (3 H, s), 2.23-2.40 (1 H, m), 1.55-1.90 (4 H, m), 1.49 (4.8 H, s), 1.45 (4.2 H, s), 1.18(3 H, d, J=6.9 Hz).

Example 18 Preparation of Compound 59

Preparation of Compound 58

Following general procedure C, compounds 57 (25 mg, 0.046 mmol, 1 eq.) and 18a (34 mg, 0.07 mmol, 1.5 eq.) were combined in the presence of trifluoroacetic acid in methylene chloride, followed by treatment with DEPC and diisopropylethylamine. The reaction mixture was separated on silica gel column in a step gradient of MeOH from 0 to 10% in CH₂Cl₂. The product was eluted by 5% MeOH and after concentration was obtained as a white solid. Yield 58: 33 mg (85%); R_(f) 0.35 (CH₂Cl₂/MeOH, 10/1); ES-MS m/z 850.7 [M+H]⁺; UV λ_(max) 215, 271 nm.

Preparation of Hydrazone 59

Compound 59 was prepared from compound 58 (33 mg, 0.04 mmol) and maleimidocaproylhydrazide (5 eq.) according to General Procedure F, reaction time—17 h. Yield 59: 17.3 mg (40%) of colorless glass; ES-MS m/z: 1057.9 [M+H]⁺; UV λ_(max) 215, 265 nm.

Example 19 Cytotoxicity Study: Compounds 29a and 39a

In vitro cytotoxicity experiments on several cell lines were performed to compare the activities of auristatin E (29a) with the ketoester 39a. The ester proved to be cytotoxic on a wide panel of cell lines, ranging from as cytotoxic as auristatin E (29a) on L2981 cells, to being approximately 17-times less cytotoxic on Daudi Burkitt's lymphoma cells (Table 4).

TABLE 4 CELL LINES USED FOR THE EVALUATION OF MAB-AURISTATIN E CONJUGATES, AND THE RELATIVE CYTOTOXIC EFFECTS OF AURISTATIN E (29a) AND THE KETOESTER DERIVATIVE 39a Antigen Expression (MFI) IC₅₀(nM) Cell Line Tumor Type LeY CD40 CD30 29a 39a L2987 lung 620 171 7 1.0 0.9 Kato III gastric 94 neg. neg. 0.7 5 SKBR3 breast 2985 20 88 1.5 6 AU565 breast 869 19 57 1.5 6 Daudi Burkitt's lymphoma 37 72 neg. 0.9 15 L428 Hodgkin's lymphoma 74 16 76 1.5 13 L540 Hodgkin's lymphoma 99 0 223 1.5 16 IM-9 Multiple myeloma 132 47 40 3.5 50 Karpas ALCL 59 1 246 1.2 13

To obtain the results shown in Table 4, the cells were exposed to drugs for 1-2 hours, and the cytotoxic effects were determined 72 hours later by the incorporation of ³H-thymidine into DNA. Antigen expression was determined by flow cytometry and is expressed in terms of mean fluorescence intensity (MFI).

Further studies were undertaken to explore the stability of 39a in human serum, and it was shown that there was no detectable hydrolysis after 120 hours incubation at 37° C. Furthermore, prolonged exposure of 39a to purified esterases from human, rhesus monkey, guinea pig and rabbit livers failed to demonstrate any conversion of 39a to auristatin E (29a). Thus it appears that 39a may not be an auristatin E prodrug, but instead exhibits activity as a benzylic ester.

Example 20 Cytotoxicity Study: Compounds 29a, 39a, 32, 33, 47, 49, 50, 52 and 58

In vitro cytotoxicity experiments were performed to compare the activities of auristatin E (29a) and drugs of this invention. 3396 and Karpas cells were exposed to the drug for 1 and 2 hr respectively. The cells were washed, and cytotoxicity was determined 96 hr later using XTT to measure mitochondrial integrity. The results are shown in Table 5.

TABLE 5 THE RELATIVE CYTOTOXIC EFFECTS OF AURISTATIN E (29a) AND DERIVATIVES Tumor IC₅₀ (nM) Cell Line Type 29a 39a 32 33 47 49 50 52 58 3396 breast 6.0 16 4.5 70 0.35 34 3.0 20 2.5 Karpas 299 ALCL 1.2 4 0.075

Example 21 Structural Effects on Hydrazone Hydrolysis

In order to analyze the structural requirements for efficient hydrazone hydrolysis under mildly acidic conditions, a series of norephedrine derivatives (Table 6) related in structure to the C-terminus of a pentapeptide of the invention were prepared.

TABLE 6 COMPOUNDS TESTED FOR HYDRAZONE HYDROLYSIS KINETICS Compound R=

43 (n = 2)44 (n = 3)45 (n = 5)

46

Condensation of N-acetylnorephedrine with various ketoacids in the presence of DCC led to the formation of the corresponding ketoesters. Upon reaction with maleimidocaproylhydrazide, hydrazones were formed. HPLC was used for stability determination at pH 5 and 7.2 at 37° C. 2-Mercaptoethanol (2 eq.) was added to quench the reactive maleimide functionalities forming compounds 43-46 in situ. The mercaptoethanol adducts 43-45 were unstable at both pH 5 and 7.2 (Table 7). In contrast, the benzylic hydrazone 46 hydrolyzed quite slowly at pH 7.2, but underwent hydrolysis at pH 5 with a half-life of 5 hours. The product formed upon hydrolysis of 46 was the parent ketoester.

TABLE 7 HYDROLYSIS OF THIOETHER-MODIFIED HYDRAZONES OF NOREPHEDRIN AT 37° C. Hydrazone t_(1/2) at pH 5.0 t_(1/2) at pH 7.2 43 <2 minutes 8 hours 44 <2 minutes 2 hours 45 <2 minutes <10 minutes 46 5 hours 60 hours

Example 22 Preparation of mAb-Drug Conjugates

A solution of monoclonal antibody (mAb) (5-10 mg/mL) in phosphate buffered saline, pH 7.2, is reduced with dithiothreitol (65 eq.) at 37° C. for 45 minutes. Separation of low molecular weight agents is achieved by size exclusion chromatography on a Sephadex G25 column, and the sulfhydryl content in the mAb is determined using 5,5′-dithiobis(2-nitrobenzoic acid) as described previously (Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) “Ellman's reagent: 5,5′-dithiobis(2-nitrobenzoic acid)-a reexamination” Anal. Biochem. 94, 75-81). There are typically between 6-9 free sulhydryl groups per mAb.

To the reduced mAb is added acetonitrile (12% final vol) followed by drug-(reactive linker) 36, 38, 40a, 40b, 48, 51 or 59 in a 1 mM solution of 9:1 acetonitrile:DMSO so that the final drug concentration is 1.9-fold higher than that of the mAb sulfhydryl groups. After 60 min at room temperature, glutathione (1 mM final concentration) and oxidized glutathione (5 mM final concentration) are added, and the solution is allowed to stand for an additional 10 min. Unbound drug is removed by repeated ultrafiltration (Amicon, YM30 filter) until there is no evidence of low molecular weight agents in the filtrate. The concentrated protein is gel filtered on Sephadex G25, a small portion of polystyrene beads is added to the pooled protein fraction, and the solution is sterile filtered using 0.4 micron filters. The protein concentration is determined at 280 nm (E_(0.1%) 1.4).

Free drug concentration is determined by reversed-phase chromatography against standard solutions of drug-(reactive linker) standards (C₁₈ Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN in 5 mM ammonium phosphate buffer, pH 7.0, from 10 to 90% in 10 min followed by 90% MeCN for 5 min at flow rate 1 mL/min). Typically, there is less than 0.5% free drug in the purified conjugate preparations. Bound drug is quantified by hydrolyzing the linked drugs from the mAb with 1 volume of pH 1.5 aqueous HCl buffer for 15 minutes at room temperature, neutralizing the solution with borate buffer at pH 8, and analyzing drug concentration using reversed phase HPLC as indicated above. There are typically between 4-7 drugs/mAb.

Example 23 Hydrolysis of Thioether-Modified Linkers and Immunoconjugates

Synthesis of Thioethers of Compounds 36, 38, 40a, 40b, 48, 51, 53 and 59

Mercaptoethanol adducts of compounds 36, 38, 40a, 40b, 48, 51, 53 and 59 were prepared in situ by a reaction of the corresponding maleimido hydrazones (0.3 mM in 10% aqueous PBS, 10% DMSO) with 2 eq. of mercaptoethanol for 15 min at room temperature. HPLC analysis (C₁₈ Varian Dynamax column, 5μ, 100 Å, linear gradient of MeCN from 10 to 90% in 5 mM ammonium phosphate buffer, pH 7.0, in 10 min, then 90% of MeCN for 5 min at flow rate 1 mL/min) showed new peaks with retention times of 0.3-1 min less then for parent hydrazones. Reaction mixtures were directly used for hydrolysis kinetics.

Kinetics of the Hydrazone Bond Hydrolysis for Thioethers of Compounds 36, 38, 40a, 40b, 48, 51, 53 and 59

Thioethers solutions (0.3 mM in 10% aqueous PBS, 10% DMSO) were adjusted to a final salt concentration of 30 mM with either 100 mM NaOAc, pH 5.0, or 100 mM Na₂HPO₄, pH 7.2, then incubated at 37° C. in a temperature controlled autosampler of the HPLC system. Aliquots (10 μL) were taken at timed intervals and the disappearance of starting material was monitored by HPLC at 215 nm (C₁₈ Varian Dynarnax column, 5μ, 100 Å, linear gradient from 10 to 90% of MeCN in 5 mM ammonium phosphate buffer, pH 7.0, in 10 min, then 90% of MeCN for 5 min at flow rate 1 mL/min). The results were expressed as percents of starting hydrazones. Half-lives of hydrazones were calculated from these curves and are listed in Table 8.

Immunoconjugate Hydrolysis Kinetics

Conjugates (1 mg/mL) in 10% aqueous DMSO, 50 mM buffer (either NaOAc, pH 5.0, or 50 mM Na₂HPO₄, pH 7.2) were incubated at 37° C. in a temperature controlled autosampler of the HPLC system. Aliquots (100 μL) were taken at timed intervals and the formation of a drug (keto-ester) was monitored by HPLC at 250 nm (C₁₈ Varian Dynamax column, 5μ, 100 Å, linear gradient from 10 to 90% of MeCN in 5 mM ammonium phosphate buffer, pH 7.0, in 10 min, then 90% of MeCN for 5 min at flow rate 1 mL/min). Free drug was quantitated by integrating the corresponding peak (positively identified by comparison to a standard). The results were presented as percents of total drug released. Half-lives of immunoconjugates were calculated from these curves and are listed in Table 8.

TABLE 8 HYDROLYSIS OF THIOETHER-MODIFIED LINKERS AND IMMUNOCONJUGATES AT 37° C. Conjugate t_(1/2) at pH 5.0 t_(1/2) at pH 7.2 Thioether of 38 4 hours 9 hours Thioether of 40a 8 hours 110 hours Thioether of 48 3.5 hours >50 hours Thioether of 51 3 hours >40 hours Thioether of 53 2 hours <30 hours Thioether of 59 4.5 hours >55 hours BR96-S-36 50 hours >500 hours BR96-S-40a 15 hours 250 hours BR96-S-48 15 hours >150 hours BR96-doxorubicin 3.5 hours >120 hours AC10-S-48 17 hours >350 hours

Example 24 Preparation of hBR96-S-42 Conjugate

To 450 μL of PBS, pH 7.2, containing 3.0 mg hBR96 was added 50 μL of 100 mM dithiothreitol (DTT) in water. The mixture was incubated at 37° C. for 30 min. Excess DTT was removed from the reduction reaction by elution through a PD10 column (Pharmacia) with PBS containing 1 mM DTPA. The number of free thiols per antibody was determined to be 8.5, by measuring the protein concentration (A₂₈₀, assuming the absorbance of a 1 mg/mL solution=1.4) and the A₄₁₂ of an aliquot of the protein treated with DTNB, assuming a molar extinction coefficient of 14,150.

The pH of the 1.2 mL reduced antibody solution was raised to 8.7 by the addition of 150 μL 500 mM Na-Borate/500 mM NaCl, pH 8.7. A solution containing a 12-fold excess of compound 42 over antibody was prepared in 150 μL DMSO. The solution of compound 42 was added to the reduced antibody solution with vigorous stirring and the reaction mixture incubated at room temperature for about 3 hours. The reaction mixture was quenched by the addition of sufficient 200 mM sodium tetrathionate to make the solution 1 mM.

The quenched reaction mixture was purified by immobilization on an ion exchange matrix, rinsing with a partial organic solvent solution, elution from the matrix and buffer exchange into PBS. Thus, the quenched reaction mixture was diluted to 35 mL in 25 mM Tris, pH 9 (“Eq. Buffer”), then loaded onto an EMD TMAE column equilibrated in Eq. Buffer. The immobilized conjugate was rinsed with a mixture of 20% acetonitrile/80% Eq buffer, then eluted with 0.5 M NaCl/19 mM Tris, pH 9. Fractions were analyzed by size exclusion chromatography and pooled. The eluted conjugate was concentrated by centrifugal ultrafiltration, then eluted through a PD 10 column in PBS, producing 1.2 mL of a 2.0 mg/mL solution (based on A₂₈₀). Elution of conjugate through a C₁₈ column indicated that any unconjugated drug was below the detection level (˜0.2 μM). Treatment of the conjugate with pH 1.7 buffer for 15 min, followed by C₁₈ HPLC analysis indicated that the drug was covalently conjugated to the antibody. Comparison of the HPLC peak area from the hydrolyzed conjugate with an standard curve of 39a determined the level of the drug attached to a given quantity of conjugate; comparison with the antibody concentration determined by A₂₈₀, gave a ratio of 3.2 drug/hBR96. Treatment of H3396 cells with this conjugate inhibited cell growth with an IC₅₀ of about 1.5 μg/mL.

Example 25 In Vitro Cytotoxicity Data for mAb-S-36

The cytotoxic effects of the mAb-S-36 conjugates on L2987 human lung adenocarcinoma cells (strongly BR96 antigen positive, weakly S2C6 antigen positive) and on Kato III human gastric carcinoma cells, (strongly BR96 antigen positive, S2C6 antigen negative) are shown in FIGS. 18A, 18B and 18C. These figures show the in vitro cytotoxicity of drugs and mAb-drug conjugates on (FIG. 18A) and (FIG. 18B) L2987 human lung adenoma cells, and (FIG. 18A) and (FIG. 18C) Kato III human gastric carcinoma cells. L2987 cells are positive for the antigens recognized by BR96 (LeY) and S2C6 (CD40), but express the LeY antigen at higher levels. Kato III cells are positive for the LeY antigen and negative for CD40. Cells were exposed to the conjugates for 2 hours, washed, and the cytotoxic effects were determined 3 days later using a thymidine incorporation assay.

Auristatin E (29a) and the ketone derivative 35 were at least 100-fold more cytotoxic than doxorubicin on L2987 and Kato III cells (FIG. 18A). On both cell lines, the BR96-S-36 conjugate displayed increased potency compared to S2C6-S-36, suggesting some degree of immunological specificity. The low potency of BR96-S-36 is most likely due to its stability at pH 5 (Table 8), suggesting that only a small portion of the conjugated drug is released under the assay conditions during a two day period.

Example 26 In Vitro Cytotoxicity Data for mAb-S-40a

The cytotoxic effects of specific conjugates on several cell lines are shown in FIGS. 19A and 19B. These figures show the cytotoxic effects of auristatin E and auristatin E-containing conjugates on L2987 human lung adenocarcinoma cells (FIG. 19A); and various hematologic cell lines treated with AC10-S-40a (FIG. 19B). In all assays, cells were exposed to the drugs for 2 hours, washed, and the cytotoxic activities were measured 72 hours later using ³H-thymidine incorporation.

On L2987 cells (FIG. 19A), BR96-S-40a was significantly more cytotoxic than S2C6-S-40a, reflecting increased LeY antigen expression compared to CD40 (Table 4). The effects of the AC10-S-40a conjugate on several hematologic cell lines are shown in FIG. 19B. The most sensitive cells were L540 Hodgkin's lymphoma and Karpas anaplastic large cell lymphoma, both of which strongly express the CD30 antigen. The L428 cell line, which is intermediate in CD30 antigen expression, was affected to a lesser extent by the AC10-S-40a conjugate. The effects appeared to be immunologically specific, since the antigen-negative control cell line, Daudi, was quite insensitive to the conjugate. One of the interesting findings in this study was that IM-9, a strong expresser of the CD30 antigen (Table 4), was also insensitive to the AC10-S-40a conjugate. Upon further analysis, it was found that unlike the other cell lines, IM-9 did not internalize bound conjugate. Thus, these results demonstrate that activity requires not only specific binding, but also antigen internalization.

Example 27 In Vivo Activities of mAb-S-40a Conjugates

Nude mice with subcutaneous L2987 human lung adenocarcinoma or 3396 human breast carcinoma xenografts were injected with conjugates or drug according to the schedule shown in FIGS. 20A and 20B. The BR96-S-40a conjugates bound to the tumor lines, while the IgG-S-40a did not. All animals were monitored daily for general health, and every 3-8 days for weight and tumor growth. Tumor volumes were estimated using the formula: (longest dimension)×dimension perpendicular²/2. There was no toxicity associated with the mAb-S-40a conjugates. The effects were compared to those of auristatin E at the maximum tolerated dose. Estradiol implants were used to sustain the growth of 3396 tumors. The implants released estradiol over a period of 60 days, at which point the experiment was terminated.

Example 28 In Vitro Cytotoxicity Data for mAb-S-40A, mAb-S-48, mAb-S-51, and mAb-S-59

The cytotoxic effects of the conjugates on H3396 human breast carcinoma cells (strongly BR96 antigen positive) are shown in FIG. 21. To obtain this data, L2987 cells in RPMI medium were plated into 96-well plates (5,000 cells/well), and after 24 hours at 37° C., various concentrations of the conjugates in medium (0.05 mL) were added to triplicate samples. After 1 hour at 37° C., the cells were washed, and incubation was continued for an additional 24 hours, at which time the cells were washed again. The cytotoxic effects were determined after an additional 3 days using XTT as an indicator of cell viability. The data show that while all the conjugates are highly cytotoxic to H3396 cells, the BR96 conjugates prepared from compounds 48, 51 and 59 are more potent then BR96-40a.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference. For example, the book in Comprehensive Organic Transformations, A Guide to Functional Group Preparations, Second Edition, Richard C. Larock, John Wiley and Sons, Inc., 1999, and particularly the references cited therein, is incorporated herein by reference for all purposes.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. 

1. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is-hydrogen or lower alkyl; and R⁹ is

R¹¹ is hydrogen or lower alkyl; R¹² is lower alkyl, halogen, or methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and

wherein: R¹⁴ is a direct bond, lower alkylene, arylene, or arylene(lower alkylene); R¹⁵ is hydrogen, lower alkyl or aryl; R¹⁶ is arylene(lower alkylene), lower alkylene, arylene, aryl (lower alkylene), or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and


2. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹ is hydrogen.
 3. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹ and R² are methyl.
 4. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R³ is isopropyl.
 5. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle and R⁵ H or methyl.
 6. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁴ is lower alkyl, and R⁵ is H or methyl.
 7. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or
 6. 8. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁶ is lower alkyl.
 9. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁸ is hydrogen.
 10. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁹ is


11. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 10 wherein R¹⁰ is


12. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 10 wherein R¹⁰ is


13. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 10 wherein R¹⁴ is arylene or lower alkylene; R¹⁵ is lower alkyl or aryl; and R¹⁶ is lower alkylene.
 14. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁹ is


15. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 14 wherein R¹⁰ is


16. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 14 wherein R¹⁰ is


17. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 14 wherein R¹⁴ is arylene or lower alkylene; R¹⁵ is lower alkyl or aryl; and R¹⁶ is lower alkylene.
 18. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁹ is

 and R¹³ is


19. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 18 wherein R¹⁵ is lower alkyl; and R¹⁶ is lower alkylene.
 20. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁹ is


21. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 20 wherein R¹⁶ is lower alkylene or arylene.
 22. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R⁹ is


23. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 22 wherein R¹⁶ is lower alkylene or arylene.
 24. A compound of claim 1 having the structure

or a pharmaceutically acceptable salt or solvate thereof.
 25. A compound of claim 1 having the structure

or a pharmaceutically acceptable salt or solvate thereof wherein R⁴is iso-propyl or sec-butyl.
 26. A compound of claim 1 having the structure

or a pharmaceutically acceptable salt or solvate thereof.
 27. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹⁷ is


28. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹⁷ is


29. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹⁷ is


30. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹⁷ is

and Y═O or S.
 31. A compound of claim 1 having the structure

or a pharmaceutically acceptable salt or solvate thereof.
 32. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 1 wherein R¹³ is


33. A compound of claim 1 having the structure

or a pharmaceutically acceptable salt or solvate thereof.
 34. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is hydrogen or lower alkyl; R¹¹ is hydrogen or lower alkyl; R¹² is lower alkyl, halogen, or methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and R²⁰ is

wherein: R¹⁴ is a direct bond, arylene(lower alkylene), lower alkylene or arylene; R¹⁵ is hydrogen, lower alkyl or aryl; R¹⁶ is lower alkylene, arylene(lower alkylene), arylene, or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is an activated ester, an anhydride, an acid chloride, an isocyanate, an isothiocyanate,

wherein X is a leaving group.
 35. A compound of claim 34 having the formula

or a pharmaceutically acceptable salt or solvate thereof.
 36. A compound of claim 34 having the formula

or a pharmaceutically acceptable salt or solvate thereof wherein R⁴is iso-propyl or sec-butyl, and R⁵ is hydrogen.
 37. A compound of claim 34 having the formula

or a pharmaceutically acceptable salt or solvate thereof.
 38. A compound of claim 34 having the formula

or a pharmaceutically acceptable salt or solvate thereof.
 39. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 34 wherein R¹⁷ is isocyanate, isothiocyanate,

wherein X is a leaving group.
 40. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is hydrogen or lower alkyl; R¹¹ is hydrogen or lower alkyl; R¹² is lower alkyl, halogen, or methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; R¹⁶ is arylene(lower alkylene), lower alkylene, arylene, or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is an activated ester, an anhydride, an acid chloride, an isocyanate, an isothiocyanate,

wherein X is a leaving group.
 41. A compound of claim 40 having the formula

or a pharmaceutically acceptable salt or solvate thereof.
 42. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 40 wherein R¹⁷ is isocyanate, isothiocyanate,

wherein X is a leaving group.
 43. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is hydrogen or lower alkyl; R¹¹ is hydrogen or lower alkyl; R¹² is lower alkyl, halogen, or methoxy, and m is 0-5 where R¹² is independently selected at each occurrence; and where R²⁰ is

wherein: R¹⁴ is a direct bond, arylene(lower alkylene), lower alkylene or arylene; R¹⁵ is hydrogen, lower alkyl or aryl; R¹⁶ is arylene(lower alkylene), lower alkylene, arylene, or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is an activated ester, an anhydride, an acid chloride, an isocyanate, an isothiocyanate,

wherein X is a leaving group.
 44. A compound of claim 43 having the formula

or a pharmaceutically acceptable salt or solvate thereof.
 45. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 43 wherein R¹⁷ is isocyanate, isothiocyanate,

wherein X is a leaving group.
 46. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is hydrogen or lower alkyl; R¹¹ is hydrogen or lower alkyl; R¹² is lower alkyl, halogen, or methoxy, and m is 0-5 where R12 is independently selected at each occurrence; R¹⁶ is arylene(lower alkylene), lower alkylene, arylene, or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is an activated ester, an anhydride, an acid chloride, an isocyanate, an isothiocyanate,

wherein X is a leaving group.
 47. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 46 wherein R¹⁷ is isocyanate, isothiocyanate,

wherein X is a leaving group.
 48. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is hydrogen or lower alkyl; and R²⁰ is

wherein: R¹⁴ is a direct bond, arylene(lower alkylene), lower alkylene or arylene; R¹⁵ is hydrogen, lower alkyl or aryl; R¹⁶ is arylene(lower alkylene), lower alkylene, arylene, or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is an activated ester, an anhydride, an acid chloride, an isocyanate, an isothiocyanate,

wherein X is a leaving group.
 49. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 48 wherein R¹⁷ is isocyanate, isothiocyanate,

wherein X is a leaving group.
 50. A compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, wherein, independently at each location: R¹ is hydrogen or lower alkyl; R² is hydrogen or lower alkyl; R³ is lower alkyl; R⁴ is lower alkyl, aryl, or —CH₂—C₅₋₇carbocycle when R⁵ is H or methyl, or R⁴ and R⁵ together form a carbocycle of the partial formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently hydrogen or lower alkyl and n is 2, 3, 4, 5 or 6; R⁶ is hydrogen or lower alkyl; R⁷ is sec-butyl or iso-butyl; R⁸ is hydrogen or lower alkyl; and R²⁰ is

wherein, R¹⁵ is hydrogen or lower alkyl, R¹⁶ is arylene(lower alkylene), lower alkylene, arylene, or —(CH₂OCH₂)_(p)CH₂— where p is 1-5; and R¹⁷ is an activated ester, an anhydride, an acid chloride, an isocyanate, an isothiocyanate,

wherein X is a leaving group.
 51. A compound or a pharmaceutically acceptable salt or solvate thereof of claim 50 wherein R¹⁷ is isocyanate, isothiocyanate,

wherein X is a leaving group.
 52. A composition comprising a compound or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1-23, 24-26, and 27-33 and a pharmaceutically acceptable carrier, diluent or excipient. 